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First published online 12 September 2007
doi: 10.1242/dev.011510
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Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093, USA.
* Author for correspondence (e-mail: marty{at}ucsd.edu)
Accepted 2 August 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Arabidopsis, Development, Stigma, Transmitting tract, Pollen tube growth
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During fertilization, pollen grains germinate on the stigma and grow
downward through the stigma and transmitting tract before diverging laterally
toward the ovules. Transmitting tract cells facilitate pollen tube growth by
secreting a complex extracellular matrix (ECM) rich in acidic polysaccharides,
and by undergoing a program of developmentally controlled cell death
(Lennon et al., 1998
;
Wang et al., 1996;
Crawford et al., 2007). A
number of mechanisms involving chemical gradients and/or signal molecules have
been proposed, whereby pollen tubes are guided from the stigma into the
transmitting tract and out of the transmitting tract towards the awaiting
ovules (Johnson and Preuss,
2002; Palanivelu et al.,
2003; Palanivelu and Preuss,
2006). In the absence of proper transmitting tract
differentiation, pollen tube growth is limited and fertility is reduced
(Crawford et al., 2007).
Since proper development of female reproductive tissue is essential to the
reproductive success of the plant, this process is likely to be highly
regulated. A number of genes have been identified as important for patterning
the stigma, style, septum and transmitting tract. These include
SPATULA (SPT), STYLISH1 (STY1),
STYLISH2 (STY2) and ETTIN (ETT).
SPT encodes a basic helix-loop-helix (bHLH) transcription factor
expressed early in septum and stigma development. Loss of SPT
function leads to defects in septum and apical carpel fusion, loss of
transmitting tract and a decrease in stigmatic tissue development
(Alvarez and Smyth, 2002;
Heisler et al., 2001).
STY1 and STY2 encode RING-finger proteins that function in
the development of the style (Kuusk et
al., 2002; Sohlberg et al.,
2006). spt mutants are epistatic to sty1
mutants, suggesting that SPT and STY act in the same pathway
(Sohlberg et al., 2006).
ETT, which encodes an auxin-response factor (ARF), has been shown to
restrict the expression domain of SPT. In ett mutants,
transmitting tract tissue develops on the outside of the gynoecium, and
removing SPT function from ett mutants rescues this defect
(Heisler et al., 2001).
Previous studies indicate that the hormone auxin plays an important role in
controlling development of the gynoecium. High levels of auxin have been
postulated to accumulate in the style and form a gradient downward through the
gynoecium (Nemhauser et al.,
2000). Treatment of wild-type gynoecia with the auxin transport
inhibitor NPA produces enlarged stigmas and styles reminiscent of weak
ett phenotypes. Furthermore, application of NPA to spt
gynoecia partially restores gynoecium development, indicating that auxin is
especially important in patterning the development of the female reproductive
tract (Nemhauser et al.,
2000).
In this work, we report three new genes that play an important role in the complex program of gynoecium development. HEC1, HEC2 and HEC3 encode closely related bHLH transcription factors with overlapping functionality. Loss of HEC function leads to defects in the development of the transmitting tract, septum and stigma and to a decrease in fertility. Conversely, overexpression of HEC genes causes both the production of ectopic stigmatic tissue and gain-of-function phenotypes implicating them as components of the auxin-signaling pathway. HEC proteins heterodimerize with SPT in a yeast two-hybrid system, suggesting that these proteins are likely to cooperatively interact in controlling development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). It was
genotyped using the gene-specific primers oKG156
(5'-ACCACAACAACACTTACCCTTTTC-3') and oKG157
(5'-GTTCCACACCCTTTCATAACCACT-3') to amplify a wild-type fragment
and the T-DNA-specific primer (5'-CCCATTTGGACGTGAATGTAGACAC-3';
sequence provided by GABI-KAT) in combination with oKG156.
The hec3 allele corresponds to the SALK_005294 line
(Alonso et al., 2003). It was
genotyped using primers C-X1 (5'-GTGCTATTTCGTGAAGAGACAAGAGA-3')
and C-X4 (5'-TCCTAACAAACCCTTATTTCGTATCCA-3') to amplify a
wild-type fragment and C-X4 in combination with the T-DNA-specific primer
JMLB2 (5'-TTGGGTGATGGTTCACGTAGTGGG-3').
The ett-7 allele was kindly provided by Patricia Zambryski
(University of California, Berkeley, CA). The spt-2 allele was kindly
provided by David Smyth (Heisler et al.,
2001).
Generation of transgenic plants
HEC2-RNAi lines were generated by amplifying a 180 bp fragment,
using primers oKG93 (5'-GGGATCCTCTAATGAACATGATGATGC-3') and oKG110
(5'-TTATCGATTAACCGGGTTGGTGGTGAGGCATG-3') for the 5'-3'
orientation, and primers oKG94 (5'-CCTCGAGTCTAATGAACATGATGATGC-3')
and oKG91 (5'-GGGTACCCCGGGTTGGTGGTGAGGCATG-3') for the
3'-5' orientation. Both fragments were cloned into the pHANNIBAL
vector (Wesley et al., 2001)
and the entire cassette was subsequently cloned into pART27
(Gleave, 1992) to generate
KG154-1. Phenotypes were analyzed in the T1 generation.
The HEC3 rescue construct was generated by amplifying a genomic fragment that included 2979 bp upstream of the translational start site and the coding region, using primers oKG121 (5'-CCGTCGACCTTCCCATGCCTTGTAATCAC-3') and oKG256 (5'-TGTCGACCTAGATTAATTCTCCTACTC-3'). A SalI fragment was cloned into pMX202 to generate KG153-7. KG153-7 was transformed into the hec1 hec3 double mutant and phenotypes were analyzed in the T1 and T2 generations. In situ analysis was performed to confirm that the promoter was sufficient to drive both septum and funiculus expression.
The HEC1p::HEC1:GUS construct was generated by amplifying a genomic fragment that included 2972 bp upstream of the translational start and the coding region up to, but not including, the stop codon. The region was amplified using primers oKG117 (5'-CCGTCGACCACCCAACTACCAACTAAATG-3') and oKG118 (5'-CCGTCGACTCTAAGAATCTGTGCATTGCC-3'). A SalI fragment was cloned into pBI101.1 to generate KG92-12.
The HEC2p::GUS construct was generated by amplifying a genomic fragment that included 3058 bp upstream of the translational start codon, using primers oKG119 (5'-GGGTCGACGAATACAGAGCACTTGTTCAAG-3') and oKG187 (5'-CAGATCTCCTCCTTTTTTGTGGAATTTATAG-3'). A SalI/BglII fragment was cloned into pBI101.1 to generate KG128-6.
The HEC3p::GUS construct was generated by amplifying a genomic fragment that included 2979 bp upstream of the translational start codon, using primers oKG121 (5'-CCGTCGACCTTCCCATGCCTTGTAATCAC-3') and oKG188 (5'-GGTCGACAATTTTTGTTTGTTTGGTTCG-3'). A SalI fragment was cloned into pBI101.1 to generate KG141-5.
To generate the overexpression lines, coding sequences were PCR amplified
using Col genomic DNA as a template and cloned into the pCR2.1-TOPO vector.
HEC1 was amplified using primers oKG83
(5'-GGTCGACATCTTTCTCTATGGATTCTGAC-3') and oKG84
(5'-CGGATCCCATCATCATCTAAGAATCTGTG-3'). A
SalI/BamHI fragment was cut out of pCR2.1 and cloned into
pBIN-JIT (Ferrandiz et al.,
2000) to create KG72-4. HEC2 was amplified using primers
oKG89 (5'-CGTCGACAAAAAGGAGGATGGATAACTC-3') and oKG90
(5'-CCCCGGGCATCATCATCTAAGAATCTGTG-3'). A
SalI/SmaI fragment was cloned into pBIN-JIT to create
pKG68-4. HEC3 was amplified using primers oKG95
(5'-CGTCGACCCAAACAAACAAAAATTATGA-3') and oKG96
(5'-CGGATCCCTTGTCTAGATTAATTCTCC-3'). A
SalI/BamHI fragment was cloned into pBIN-JIT to create
pKG80-22. In the pBIN-JIT vector, the coding sequences were placed under the
control of a double repeat of the 35S promoter. Overexpression phenotypes were
analyzed in the T1 and T2 generations.
The integrity of all constructs was confirmed by sequencing.
RT-PCR
RNA was isolated using the Qiagen RNeasy Plant Mini Kit. The reverse
transcriptase reaction was performed using the Promega Reverse Transcription
System. HEC1 was amplified with primers oKG83 and oKG84.
HEC2 was amplified with primers oKG89 and oKG90. HEC3 was
amplified with primers oKG95 and oKG96. A ß-TUBULIN control
amplification was performed using primers
(5'-GGACAAGCTGGGATCCAGG-3') and N-1137
(5'-CGTCTCCACCTTCAGCACC-3'). The annealing temperature was
58°C. The PCR amplification in Fig. S2A,B (see Fig. S2A,B in the
supplementary material) was carried out using 2 µl of reverse transcriptase
reaction, the HEC amplification was performed with 35 cycles, and
TUBULIN was amplified using 25 cycles. In the PCR amplification shown
in Fig. S2C (see Fig. S2C in the supplementary material), HEC2 was
amplified with 30 cycles using 1 µl of reverse transcriptase reaction,
SPT was amplified with 28 cycles using 1 µl of reverse
transcriptase reaction, and TUB was amplified with 25 cycles using
0.5 µl of reverse transcriptase reaction.
In situ hybridization
In situ hybridization was performed as described by Dinneny et al.
(Dinneny et al., 2004). An
antisense HEC1 probe was transcribed with T7 polymerase (Promega)
using a full-length coding sequence in pCR2.1 (KG62-1) that had been
linearized with HindIII. An antisense HEC2 probe was
transcribed with T7 polymerase using a full-length coding sequence in
pBluescript (KG100-1) linearized with SpeI. An antisense
HEC3 probe was transcribed with T7 polymerase using a full-length
coding sequence in pCR2.1 (KG76-2) linearized with SpeI.
Microscopy and histology
Staining for ß-glucuronidase expression was as described
(Blázquez et al., 1997)
with minor modifications. Wild-type (Columbia ecotype) and transgenic fruit
and flowers were fixed and analyzed by scanning electron microscopy as
previously described (Liljegren et al.,
2000).
Aniline Blue staining for pollen tubes was performed after emasculating
flowers just prior to pollination (late stage 12), growing them for another
18-24 hours to allow transmitting tract and ovule development to be completed,
and then hand-pollinating them maximally. After allowing another 24 hours for
pollen growth, they were fixed, cleared, stained with Aniline Blue
(Jiang et al., 2005) and
examined under a fluorescence microscope.
Staining with Alcian Blue 8GX was used to visualize the transmitting tract.
Alcian Blue stains the acidic mucopolysaccharide component of the transmitting
tract ECM (Scott and Dorling,
1965). Paraplast-embedded flowers and inflorescences were
sectioned at 4 µm and fixed to slides. Slides were then de-waxed with
Histoclear (National Diagnostics), rehydrated through a gradual ethanol
series, counterstained for 5 minutes with 0.1% Nuclear Fast Red, rinsed,
stained for 5 minutes with Alcian Blue pH 3.1, rinsed again, dried briefly at
37°C, then mounted directly in Permount (Fischer Scientific).
Yeast two-hybrid system
Directed yeast two-hybrid interactions were conducted as described
(Pelaz et al., 2001).
Full-length HEC1 was cloned into both the bait vector pBI-880 to make
SP7-2 and into the prey vector pBI-771 to make SP18. Full-length HEC2
was cloned into both the bait vector pBI-880 to make SP8-4 and into the prey
vector pBI-771 to make SP19. Full-length HEC3 was cloned into the
prey vector pBI-771 to make SP20. The full-length HEC3 in the bait
vector was able to activate the reporters on its own. Hence, a partial
HEC3 fragment, which included amino acids 92-224, was cloned into
pBI-880 to make SP14-1. The SPT prey vector had previously been isolated from
a cDNA library in a yeast two-hybrid screen with IND. The partial clone
contains amino acids 47-373.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Reasoning that bHLH proteins related to IND might also function in plant
development, we performed a BLAST search to find those genes most closely
related to IND. HEC1 (At5g67060; BHLH088), HEC2 (At3g50330;
BHLH037) and HEC3 (At5g09750; BHLH043) were thus identified. All
three HEC genes were subsequently found to function in gynoecium
development, but to have roles distinct from that of IND.
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). HEC1 and
HEC2 are the most closely related and share 61% amino acid identity across the
length of the proteins and 100% identity within the bHLH domain. Previous
phylogenetic analysis of the Arabidopsis bHLH transcription factor
family placed HEC1 and HEC2 in regions that arose from a
putative interchromosomal duplication event
(Toledo-Ortiz et al., 2003).
Protein alignment of the HECs with IND and more-distantly related bHLH factors
reveals that the HECs, like IND, lack a conserved glutamate at position 13 of
the basic domain (Fig. 1D,
asterisk). This glutamate has been shown to be important for DNA binding
(Ellenberger et al., 1994;
Ma et al., 1994). Thus, if the
HECs regulate gene transcription through DNA binding, they are likely to do so
through the use of other residues.
The HEC genes are expressed in the developing septum, transmitting tract and stigma
To investigate whether the HEC genes function in gynoecium
development, we analyzed their expression patterns using both RNA in situ
hybridization and ß-glucuronidase (GUS) reporter gene constructs. In
contrast to the valve margin expression pattern described for IND
(Liljegren et al., 2004), all
three HEC genes were found to be expressed in the stigma and septum
during stages 8 to 12 of flower development.
RNA in situ analysis showed that for all three HECs, expression was first observed during stage 8 of gynoecium development, in the medial ridges of the septum, which have grown together and fused at this time (Fig. 2A-C, large arrowheads), and in the apical tips (Fig. 2G, lower arrow), where the stigma will arise. By stage 10 to early stage 12, hybridization signal was localized to the transmitting tract (Fig. 2D-H, large arrowheads) and developing stigmas (Fig. 2G,H, arrows). Patchy signal was also apparent in the ovules for HEC1 and HEC2 (Fig. 2D-E, small double arrowheads) and in the ovule funiculus for HEC3 (Fig. 2F, arrow). By late stage 12, just prior to fertilization, HEC3 expression was still evident in the transmitting tract (Fig. 2I,J, large arrowheads) and was strong in the ovule funiculus (small arrowheads), but was no longer visible in the stigma (arrow). HEC1 and HEC2 expression could no longer be detected by late stage 12 (data not shown).
GUS reporter results confirmed the septum and stigma expression of HEC1 and HEC2 (see Fig. S1A-D in the supplementary material) and also indicated that HEC3 funiculus expression continued even after pollination (see Fig. S1E in the supplementary material, arrowheads). Some HEC2p::GUS lines also expressed GUS in pollen and the nectaries (data not shown), and a number of the HEC3p::GUS lines showed expression in vasculature (see Fig. S1E in the supplementary material, arrow). GUS analysis did not confirm HEC3 transmitting tract and stigma expression, HEC1 and HEC2 ovule expression, or HEC1 anther expression, presumably because the GUS constructs lack essential DNA regulatory elements necessary for them to represent the entire pattern of expression indicated by RNA in situ hybridization.
The close sequence similarity of the HEC genes and their overlapping expression patterns suggest that they might have partially redundant functions in stigma and septum development.
hec1 and hec3 mutations reduce fertility
To determine the functions of the HEC genes, we identified T-DNA
insertion lines in HEC1 and HEC3 from available mutant
collections (Fig. 1C) and
confirmed these lines as RNA-nulls by RT-PCR (see Fig. S2A,B in the
supplementary material). An absence of HEC3 RNA in the hec3
mutant was further demonstrated by in situ hybridization (data not shown). No
satisfactory mutants in HEC2 were available at the time of this
work.
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Defects in pollen tube growth in the hec mutants
Stigma and transmitting tract provide the apical-to-basal tissue path for
pollen tube growth in Arabidopsis. Since the HEC genes are
expressed in these tissues, it seemed likely that the loss of fertility in
hec mutants would correlate with aberrant or reduced pollen tube
growth. To visualize pollen tubes within the ovary, we used an Aniline Blue
staining technique (Jiang et al.,
2005) 24 hours after hand-pollinating emasculated carpels
(Materials and methods). Aniline Blue stains callose, a component of pollen
tubes, allowing them to be visualized by fluorescence microscopy.
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HEC1 and HEC3 are necessary for stigma and transmitting tract development
Both stigma and style showed obvious developmental abnormalities in
hec mutants. Compared with wild type
(Fig. 4K), stigmas were smaller
and more variable in size in hec1 hec3 mutants
(Fig. 4L). Although not evident
in Fig. 4, there was also a
slight tendency for the style to be somewhat longer in the double mutant. To
visualize the transmitting tract in hec mutants, post-fertilization
flowers were thin-sectioned and stained with Alcian Blue, a dye that detects
acidic polysaccharides characteristic of the transmitting tract ECM. Wild type
showed a characteristically large, intensely staining transmitting tract
(Fig. 4A,H, arrowheads). The
transmitting tract of hec1 was indistinguishable from that of wild
type in size, staining intensity and cytology
(Fig. 4B). The hec3
transmitting tract was smaller in size than wild type in both the septum and
the style (Fig. 4C,I), but had
the same general appearance as wild type. The hec1 hec3 double
mutant, however, had dramatically reduced Alcian Blue staining in both the
style and the septum compared with wild type
(Fig. 4D,E,J). In analyzing
pre-fertilization stages of development, we found that the transmitting tract
of the hec1 hec3 double mutant had no delay in onset of ECM
production, but produced less ECM than wild type (see Fig. S3 in the
supplementary material). Taken together, these data demonstrate that
HEC1 and HEC3 are redundantly required for transmitting
tract differentiation.
Reducing HEC2 RNA levels in the hec1 hec3 double mutant intensifies defects in stigma and septum development
Given the sequence similarity and overlapping expression domains among the
HEC genes, and considering the synergistic nature of hec1
and hec3 single mutations, it seemed likely that all three
HEC genes would share functionally related roles in
Arabidopsis. To confirm this hypothesis and to substantiate a role
for HEC2 in gynoecium development, we used RNAi to create the
equivalent of a hec1 hec2 hec3 triple mutant. To make the RNAi
construct we used 180 bp from the 5' coding region of HEC2
(Fig. 1C). This region contains
some areas of close sequence similarity to HEC1, but little
similarity to HEC3. The construct was transformed into both the wild
type and the hec1 hec3 double mutant.
RT-PCR analysis of several independent lines of HEC2-RNAi in the wild type revealed a strong to moderate reduction in the level of both HEC1 and HEC2 RNA and only a slight effect on HEC3 RNA (see Fig. S2C,D in the supplementary material). These HEC2-RNAi lines, equivalent to hec1 hec2 double mutants, showed little or no effect on fertility (data not shown). However, when the HEC2-RNAi construct was transformed into the hec1 hec3 double mutant, 8 of 32 lines exhibited complete sterility (Fig. 3F). Such lines had dramatic defects in apical gynoecium development, with a complete absence of stigmatic tissue and, in many cases, an incomplete fusion of the apical region of the style (Fig. 4M,N). The style of HEC2-RNAi hec1 hec3 plants was exceptionally long. We found severe effects on septum and transmitting tract development in HEC2-RNAi hec1 hec3 gynoecia (Fig. 4F,G). The septum was either unfused (Fig. 4G, arrow) or had only a few cells at its thinnest point (Fig. 4F, arrow). Alcian Blue staining of the transmitting tract was never observed. Since this phenotype is reminiscent of that reported for mutants of SPT (see below), we specifically confirmed that there was no reduction in SPT RNA in the HEC2-RNAi hec1 hec3 lines (see Fig. S2C in the supplementary material). We also confirmed that the fertility defect of HEC2-RNAi hec1 hec3 lines was female-specific by crossing HEC2-RNAi hec1 hec3 pollen onto wild-type flowers. These crosses resulted in fruit of normal length (data not shown).
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Interactions between SPATULA and the HECs
The well-studied developmental regulator SPATULA (SPT)
encodes a bHLH protein that is considerably larger than any of the HEC
proteins (373 amino acids versus approximately 230 amino acids) and is poorly
conserved with the HEC proteins (Fig.
1D). Nevertheless, SPT is expressed in both septum and
stigma during stages 6 to 11 (Heisler et
al., 2001), and genetic studies have shown it to be required for
septum, transmitting tract and stigma formation
(Alvarez and Smyth, 2002). We
therefore investigated possible interactions between SPT and the
HECs.
Since SPT expression is detectable at earlier stages of gynoecium
development than is HEC expression
(Heisler et al., 2001), the
possibility existed that SPT might be a transcriptional regulator of
the HECs. We examined this by analyzing the expression of HEC1,
HEC2 and HEC3 in early carpels of plants carrying the strong
spt-2 allele (Fig.
5A-C). All three HECs continued to be expressed in this
mutant background, indicating that a functional SPT protein is not required
for HEC gene expression.
A more likely possibility was that the SPT and HEC proteins might interact
cooperatively to regulate development. bHLH proteins are known to both
homodimerize and heterodimerize, and dimer formation is essential for
transcriptional regulation (Murre et al.,
1994; Massari and Murre,
2000). We therefore used a yeast two-hybrid system to investigate
protein-protein interactions among the HEC1, HEC2, HEC3 and
SPT gene products. The HEC proteins do not form either homodimers or
heterodimers in yeast, but each is capable of heterodimerizing with SPT
(Fig. 5G,H). If the HEC
proteins function as transcriptional regulators, the data strongly suggest
that SPT is likely to be a required partner.
ETTIN is a negative regulator of HEC gene expression
Since both SPT and the HEC genes are required for aspects
of interior carpel development, specifically septum and transmitting tract
development, it is relevant that mutants in the ARF factor ETT
display a dramatic phenotype in which transmitting tract tissue develops on
the outside of the gynoecium. This externalization of transmitting tract
derives at least in part from the unrestricted expression of SPT in
carpel valves (Heisler et al.,
2001). We wanted to determine whether the HEC genes might
also be under ETT control and play a similar role in the formation of
external ectopic transmitting tract in ett mutants. HEC
expression was examined in ett-7 gynoecia and found to be equivalent
to that seen for SPT. The HECs were ectopically expressed on
the outside of ett-7 gynoecia
(Fig. 5D-F). ETT
therefore negatively regulates HEC expression in the abaxial
gynoecium in a similar manner as it does SPT.
Overexpression of the HEC genes produces ectopic stigmatic tissue, ett-like and pin-like phenotypes
To further examine the effects of ectopic HEC activity, we
generated overexpression lines in which HEC gene expression was
driven from the constitutive 35S promoter. Overexpression of each HEC
gene resulted in flowers that produced ectopic carpelloid tissue, most often
stigmatic tissue (Fig. 6B,C,D,
arrowheads; Fig. 6B,C, insets).
The data indicate that the HEC genes are able to activate carpel
identity factors when ectopically expressed.
The overexpression of HEC1 and HEC3 also occasionally led
to the production of gynoecia with defects in apical-basal polarity
reminiscent of a weak ett phenotype. Carpels had enlarged stigmas,
reduced ovaries and elongated gynophores
(Fig. 6G-I). The HEC
genes thus could be involved in the ETT-mediated auxin-signaling
pathway needed for apical-basal development. This possibility is further
supported by even more extreme phenotypes seen among overexpressing lines,
such as those shown in Fig.
6K,L. Here, pin-shaped inflorescences or carpelloid stalks were
observed, resembling those that result from loss of the auxin efflux carrier
PIN-FORMED1 (PIN1) or from treatment with the chemical NPA,
an auxin transport inhibitor (Okada et
al., 1991). These dramatic phenotypes suggest that in these
35S::HEC1 and 35S::HEC3 lines there was an alteration of
auxin levels, auxin transport and/or auxin perception.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The relationship between the HEC/IND subfamily and the SPT subfamily of bHLH transcription factors
The HEC proteins and the previously characterized valve margin
specification factor IND (Liljegren et
al., 2004) belong to an atypical group of bHLH transcription
factors. Most Arabidopsis bHLH proteins are thought to have evolved
from an ancestral group of bHLHs common to both plants and animals (group B)
and contain a conserved glutamate in the basic region
(Heim et al., 2003;
Toledo-Ortiz et al., 2003).
This glutamate contacts DNA at the bHLH recognition sequence, the E-box
(Ellenberger et al., 1994). SPT
contains this crucial glutamate, but the HECs and IND have an alanine
substitution (Fig. 1D). Animal
group-C bHLH proteins, which also lack the conserved glutamate, have been
shown to bind DNA in combination with group-B bHLHs using a different
recognition site (Bacsi et al.,
1995; Swanson et al.,
1995).
In the current study, we investigated possible interactions between
SPT and the HECs. Because SPT is expressed at
earlier stages of gynoecium development than the HEC genes
(Heisler et al., 2001), the
possibility existed that it might function as an upstream regulator of
HEC expression. We therefore examined HEC expression in
plants carrying the strong spt-2 allele and found that the
HEC genes were still expressed, implying that SPT is not
required for activation. We then considered the possibility that SPT
and HEC gene products might interact with each other. This was shown
to be the case. HEC proteins can form heterodimers with SPT in a yeast
two-hybrid system, but cannot heterodimerize or homodimerize with each other.
SPT can also heterodimerize with IND, the closest relative of the HECs (data
not shown). Since SPT is expressed more widely during development than either
the HECs or IND, but nevertheless encompasses the expression domains of both
(Heisler et al., 2001), it
seems likely that SPT and the HECs work in concert to carry out certain
developmental programs and that SPT, because of its broader expression domain,
interacts with yet other bHLH proteins to carry out additional developmental
programs. It is relevant to note here that constitutive overexpression of
SPT does not produce mutant phenotypes (M. Groszmann, PhD thesis,
Monash University, 2005), as does overexpression of the HECs. This
observation is consistent with the possibility that the HECs are able to
dimerize with broadly expressed proteins, whereas SPT requires partners with
more limited expression domains.
Do the HEC genes play a role in the auxin-signaling pathway in the gynoecium?
A fundamental role has been suggested for the hormone auxin in patterning
the Arabidopsis gynoecium, with high levels of auxin conferring
apical tissue identity (stigma/style) and low levels of auxin leading to basal
(gynophore) development (Nemhauser et al.,
2000). The ETT gene is an important mediator of auxin
effects. Mutations in ETT cause severe defects in gynoecium
development, including an enlarged stigma, an elongated gynophore and a
reduced ovary that develops transmitting tract tissue on the outside
(Sessions and Zambryski,
1995). SPT is also likely to be involved in auxin
patterning, both as a target of auxin regulation and as a mediator of auxin
effects. SPT is ectopically expressed in the ett gynoecium,
most notably in the inverted transmitting tract tissue on the outside of
carpels (Heisler et al.,
2001). Mutations in SPT can suppress mutations in
ETT (Heisler et al.,
2001). spt-2 mutants can also be partially rescued by the
auxin transport inhibitor NPA, and spt-2 gynoecia are less sensitive
than wild-type gynoecia to NPA effects on apical-basal patterning
(Nemhauser et al., 2000).
If the HECs operate coordinately with SPT as protein partners, it is likely that both proteins are targets of auxin regulation and would be similarly affected by mutations in ETT. We found that all three HEC genes were, like SPT, ectopically expressed in external transmitting tract tissue in the ett-7 mutant. The HECs, like SPT, are therefore implicated as possible targets of auxin regulation.
The overexpression phenotype of the HECs further suggests
involvement in auxin patterning. Some of the phenotypes of
HEC-overexpressing lines were similar to those of auxin-related
mutants. 35S::HEC1, 35S::HEC3 and, to a lesser degree,
35S::HEC2 lines occasionally produced gynoecia with defects in
apical-basal patterning resembling those of a weak ett mutant
(Fig. 6G-J; data not shown).
Several independent 35S::HEC1 lines produced pin-shaped, flowerless
inflorescences. We also observed stalk-like floral structures capped by
stigmatic tissue for both 35S::HEC1 and 35S::HEC3
(Fig. 6J,K). Both of the latter
phenotypes are very similar to what has been reported for the pin1
mutant (Okada et al., 1991).
PIN1 belongs to the PIN family of auxin efflux carriers,
which play an important role in setting up auxin gradients or patterns of flow
that pattern the plant (Benkova et al.,
2003; Friml, 2003;
Friml et al., 2003). The
overproliferation of stigmatic tissue in 35S::HEC lines suggests the
pooling of auxin or an increased auxin response at these sites. This
interesting link between the HEC genes and auxin should be
investigated in future studies.
In summary, the HEC genes function redundantly in patterning tissues crucial for reproductive success in the Arabidopsis gynoecium. Elucidating additional details about how the HECs interact with other carpel-patterning genes will help to provide insights into various aspects of gynoecium function, including carpel development, pollen tube growth and fertilization.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/20/3593/DC1
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