1 Department of Biology, University of North Carolina at Chapel Hill, CB #3280,
Coker Hall, Chapel Hill, NC 27599-3280, USA
2 Gene Expression Laboratory, Plant Molecular Biology, University of Lausanne,
Biology Building, 1015-Lausanne, Switzerland
3 Department of Horticultural Science, Center for Microbial and Plant Genomics,
University of Minnesota, Saint Paul, MN 55108, USA
4 Department of Biochemistry, University of Missouri-Columbia, Columbia, MO
65211, USA
5 Plant Biology Laboratory, The Salk Institute, La Jolla, CA 92186-5800,
USA
Author for correspondence (e-mail:
jreed{at}email.unc.edu)
Accepted 22 June 2005
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SUMMARY |
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Key words: Auxin response factor, ARF, Auxin, Flower maturation, Jasmonate
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Introduction |
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Transcription factors of the auxin response factor (ARF) family bind to
auxin response elements (AuxREs, 5' tgtctc 3') present in
promoters of numerous auxin-regulated genes, and ARFs mediate auxin-induced
gene expression responses (Hagen and
Guilfoyle, 2002; Liscum and
Reed, 2002
). Of the 22 predicted ARF proteins encoded in the
Arabidopsis genome, five (MP/ARF5, ARF6, NPH4/ARF7, ARF8 and ARF19)
have a glutamine-rich middle domain, and each of these can activate
auxin-induced genes in transient expression assays
(Ulmasov et al., 1999a
;
Wilmoth et al., 2005
).
Mutations in MP/ARF5, NPH4/ARF, ARF8 and ARF19 decrease
auxin gene induction responses and cause auxin-related developmental defects
at various stages of development. mp/arf5 mutants have defects in
embryonic, vascular and floral patterning
(Aida et al., 2002
;
Berleth and Jürgens, 1993
;
Hardtke and Berleth, 1998
;
Hardtke et al., 2004
;
Przemeck et al., 1996
).
nph4/arf7 mutants have defects in tropic growth of roots and
hypocotyls, and nph4/arf7 arf19 double mutants make very few lateral
roots and have small leaves (Harper et
al., 2000
; Liscum and Briggs,
1996
; Okushima et al.,
2005
; Stowe-Evans et al.,
1998
; Watahiki and Yamamoto,
1997
; Wilmoth et al.,
2005
). Light-grown arf8-1 mutant seedlings had elongated
hypocotyls (Tian et al.,
2004
). Auxin regulates glutamine-rich ARF activity by promoting
turnover of Aux/IAA proteins, which can interact with ARFs and inhibit gene
induction (Gray et al., 2001
;
Kim et al., 1997
;
Tatematsu et al., 2004
;
Tian et al., 2003
;
Tian et al., 2002
;
Tiwari et al., 2003
;
Tiwari et al., 2001
;
Ulmasov et al., 1997
;
Zenser et al., 2001
).
Gain-of-function mutations in several different IAA genes encoding
Aux/IAA proteins decrease auxin-induced turnover of the corresponding proteins
and cause phenotypes similar to those of loss-of-function arf mutants
(Reed, 2001
).
Phylogenetic analyses of Arabidopsis ARF proteins show that ARF6
and ARF8 form a clade and therefore may have overlapping functions
(Remington et al., 2004). We
have isolated plants with mutations in the ARF6 and ARF8
genes, and characterized phenotypes of single and double mutants.
arf6 and arf8 single mutant plants have delayed stamen
development and decreased fecundity, whereas arf6 arf8 double mutant
plants have a complete block in flower maturation. Decreased jasmonic acid
(JA) production caused some aspects of this phenotype.
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Materials and methods |
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Transgenic plants and genetics
For complementation, we cloned a 12.5 kb BamHI genomic DNA
fragment containing the complete open reading frame of ARF6
(T4K22.6/At1g30330) from BAC T4K22 into pCAMBIA1300. This fragment extends
3262 bp upstream of the ARF6 start codon and 5342 bp downstream of
the stop codon, and also contains the C-terminal part of one other annotated
open reading frame (predicted protein T4K22.7/At1g30320). We transformed the
T-DNA carrying this construction from Agrobacterium strain GV3101 into
arf8-3 single mutant plants by vacuum infiltration
(Bechtold et al., 1993), and
identified six transformants whose self-progeny segregated 3:1 for hygromycin
resistance encoded on the T-DNA. We fertilized each of these plants with
arf6-2 arf8-3 double mutant pollen (obtained after jasmonic acid
treatment of mutant buds). Hygromycin-resistant F1 progeny of these crosses
were allowed to self-fertilize, and the resulting F2 seed assayed for
phenotype as presented in Table
3.
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To make ARF6 and ARF8 promoter::GUS constructs, sequences
up to and including the natural start codons were amplified by PCR and cloned
upstream of the GUS start codon in a modified pPZP211 vector that
contained GUS-nos and some upstream restriction sites derived from pEBGUS
(Hagen et al., 1991). For
ARF6, primers
5'-GCTTAAGAATTAGCTGCAGAAACAAATGCTAGTTG-3'
(PstI site underlined) and
5'-CATGAGGTTGAGGATCCAACCCAGCTGAAG (BamHI site
underlined) amplified a fragment including 2043 bp upstream of the start
codon. For ARF8, primers
5'-GATTGCGACGTACTGCAGGATATTACCATCG-3' (PstI
site underlined) and 5'-CACCTTCATGACCCTGTCGACCCAATCC-3'
(SalI site underlined) amplified a fragment that included 2387 bp
upstream of the start codon. Constructs were transformed into ecotype Columbia
plants. Multiple lines were analyzed and had similar staining patterns.
Fig. 4 shows results from a
representative line.
Phenotypic analyses
Flower buds and dissected flower organs were measured using a camera lucida
attachment on a dissecting microscope. For scanning electron microscopy, buds
and flowers were fixed and processed as described
(Laux et al., 1996), except
that the tissue was dehydrated in an ethanol series (30%, 50%, 70%, 95%, 100%)
instead of an acetone series. The specimens were imaged in a Cambridge S200
scanning electron microscope (LEO Electron Microscopy, Thornwood, NY) operated
at 20 kV. Secondary electron images were acquired digitally using a 4pi image
acquisition system (4pi Analysis, Durham NC).
For rescue of anther dehiscence, 4-5 µl of linolenic acid [9(Z), 12(Z), 15(Z) octadecatrienoic acid, Cayman Chemical Company, Ann Arbor, MI; 25% stock solution in ethanol diluted to 0.1% in 0.1% Tween-20], OPDA (12-oxo-phytodienoic acid, Cayman Chemical Company, 100 mg/ml stock solution in ethanol diluted to 100 µM in 0.1% Tween-20) or JA (Sigma, 100 µM or 500 µM solution in 1% methanol 0.1% Tween-20) were applied to each flower bunch, and flowers observed 2-3 days later.
Gene expression analyses
Total RNA was isolated from frozen tissues of 8-day-old seedlings or
long-day-grown adult plants using Trizol reagent (Invitrogen Life
Technologies, Carlsbad, CA). RNA gel blot hybridizations were performed as
described (Nagpal et al.,
2000). ARF6 and ARF8 probes were made from PCR
products spanning ARF6- and ARF8-coding regions (amplified
from cDNAs). For auxin and JA induction experiments, plants were sprayed with
10 µM IAA or 500 µM JA (in 1% methanol, 0.05% Tween-20) or with buffer
alone, using a Preval sprayer (Precision Valve Corporation, Yonkers, NY). Gene
probes were as described (Stintzi and
Browse, 2000
; Tian et al.,
2002
) and were labeled with 32P by random priming.
Microarray gene expression analyses
Flower tissue was collected from Arabidopsis thaliana ecotype
Columbia plants, arf6-2/arf6-2 ARF8/arf8-3 plants, and arf6-2
arf8-3 plants. Plants were grown for 6 weeks under a 16-hour light:8-hour
dark regime. For the developmental time course, flowers were separated into
stage 1-10 flowers, stage 11-12 flowers and stage 13-14 flowers. For auxin
induction experiments, flower bunches containing flowers from stage 1 to stage
14 were used.
Tissue from approximately 40 plants was pooled for each RNA isolation and
RNA from three biological replicates was pooled for cDNA synthesis. Total RNA
(7 µg) was used to synthesize cDNA. A custom cDNA kit (Life Technologies,
Grand Island, NY) was used with a T7-(dT)24 primer for this
reaction. Biotinylated cRNA was then generated from the cDNA reaction using
the BioArray High Yield RNA Transcript Kit (Enzo Diagnostics, Farmingdale,
NY). The cRNA was then fragmented in fragmentation buffer [5x
fragmentation buffer: 200 mM Tris-acetate (pH 8.1), 500 mM KOAc, 150 mM MgOAc]
at 94°C for 35 minutes before the chip hybridization. Fragmented cRNA (15
µg) was then added to a hybridization cocktail (0.05 µg/µl fragmented
cRNA, 50 pM control oligonucleotide B2, BioB, BioC, BioD and
cre hybridization controls, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml
acetylated BSA, 100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% Tween 20).
cRNA (10 µg) was used for hybridization in a volume of 200 µl per slide.
ATH1 arrays (Redman et al.,
2004) (Affymetrix, Santa Clara, CA) were hybridized for 16 hours
at 45°C in the GeneChip Hybridization Oven 640 (Affymetrix). The arrays
were washed and stained with R-phycoerythrin streptavidin in the GeneChip
Fluidics Station 400 (Affymetrix) using wash protocol Eukge-ws2 version 4, and
arrays were scanned with the Hewlett Packard model 2500 GeneArray Scanner.
Affymetrix GeneChip Microarray Suite 5.0 software was used for washing,
scanning and basic analysis. Data were scaled to a default target intensity of
500 before importing into Genespring 5.0 software. Sample quality was assessed
by examination of 3' to 5' intensity ratios of certain genes.
Analysis of the Affymetrix gene chip data was carried out using Genespring 5.0 software. Raw data from each chip was normalized to the 50th percentile of measurements taken from that chip and genes were normalized to the median value. Following normalizations, pairwise comparisions of fold changes were carried out. For the auxin treatment experiment, data was restricted such that only genes with a raw data value greater than 100 and with an Affymetrix flag call of Present were considered. For the developmental time course experiment, data was restricted such that only genes with a raw data value greater than 300 and with a flag call of Present were considered. These raw cut-off levels were chosen as the expression level of genes below these numbers frequently had an Affymetrix flag call of Absent. Lists of genes presented in the supplementary data tables were derived by applying filter functions for threshold fold changes in gene expression. Gene expression data was also grouped using a self-organizing map using Genespring default parameters, with qualitatively similar conclusions.
We compared our microarray data to recently released data for floral stages
9, 12 and 15
(http://www.weigelworld.org/resources/microarray/AtGenExpress)
(Schmid et al., 2005). In that
dataset, among genes whose expression was referred to as `present' and with a
raw data value of at least 100, we identified 3141 genes with at least
2.5-fold differential expression at different stages. These genes included
1420 (83%) of the 1715 differentially expressed genes we identified. Taking
into account the slightly different stages analyzed, these numbers suggest
that the datasets are broadly consistent and our data are likely to be
accurate for most genes.
The data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through the following accession numbers: GSE2847, auxin induction in wild-type and arf6 arf8 flowers (samples GSM62687-GSM62693); GSE2848, auxin response factor-mediated flower gene expression (samples GSM62694-GSM62705); GSM62687, Columbia flowers_stage 1-14 untreated; GSM62688, Columbia flowers_stage 1-14_30 minutes IAA treatment; GSM62689, Columbia flowers_stage 1-14_30 minutes mock treatment; GSM62690, arf6/arf6 ARF8/arf8 flowers_stage 1-14 untreated; GSM62691, arf6/arf6 ARF8/arf8 flowers_stage 1-14_30 minutes IAA treatment; GSM62692, arf6 arf8 flowers_stage 1-14_untreated; GSM62693, arf6 arf8 flowers_stage 1-14_30 minutes IAA treatment; GSM62694, Columbia flowers_stage 1-10; GSM62695, Columbia flowers_stage 11-12; GSM62696, Columbia flowers_stage 13-14; GSM62697, Columbia_stem; GSM62698, arf6/arf6 ARF8/arf8 flowers_stage 1-10; GSM62699, arf6/arf6 ARF8/arf8 flowers_stages 11-12; GSM62700, arf6/arf6 ARF8/arf8 flowers_stages 13-14; GSM62701, arf6/arf6 ARF8/arf8 _stem; GSM62702, arf6 arf8 flowers_stage 1-10; GSM62703, arf6 arf8 flowers_stage 11-12; GSM62704, arf6 arf8 flowers_stage 13-14; GSM62705, arf6 arf8_stem.
Hormone measurements
Flower bunches were harvested into liquid nitrogen and kept frozen until
analysis. Frozen flower tissue was stored at 80°C and transported
on solid CO2 (dry ice) to Lausanne for JA measurements and to St
Paul for IAA analysis. Jasmonic acid was measured as described
(Weber et al., 1997) with
modifications described at
http://www.unil.ch/ibpv/WWWFarmer/WWWOxylipins/Docs/method.htm.
IAA content was determined for triplicate samples weighing 50 to 110 mg FW
(frozen weight). Purification and quantification of free IAA was based on the
method described by Chen et al. (Chen et
al., 1988) with modifications. Approximately 4 ml
g1 FW extraction buffer [65% (v/v) isopropanol with 0.2 M
imidazole (pH 7)] was added to each sample tube. Three tungsten carbide beads
(3 mm; Qiagen, Valencia, CA) and
40 ng g1 FW
[13C6]IAA as an internal standard were also added to the
sample tube before homogenization for 3 minutes at 15 Hz in a Mixer Mill MM
300 (Qiagen, Valencia, CA). After incubation on ice for 1 hour, samples were
centrifuged (12,000 g for 5 minutes), and 50,000 dpm
[3H]IAA was added to the supernatant as a radiotracer. The sample
was diluted 10-fold with water and applied to a conditioned 50 mg
NH2 solid phase extraction (SPE) column (Varian, Walnut Creek,
California). To condition the columns, 500 µl of hexane, acetonitrile,
water, and 0.2 M imidazole (pH 7.0) were added sequentially followed by two
water rinses of 1500 µl each. The loaded columns were washed sequentially
with 500 µl each of hexane, ethyl acetate, acetonitrile and methanol, and
300 µl phosphoric acid. IAA was eluted in four additional 700 µl
aliquots of phosphoric acid (PA). The pooled eluate was adjusted to pH 3 with
1 M succinic acid (SA; pH 6) in a ratio of PA:SA (v/v, 60:1).
IAA was further purified on a BSA column
(Murphy, 1979;
Schulze and Bandurski, 1979
)
made by linking BSA (Promega, Madison, Wisconsin) to Affiprep-10 (BioRad,
Hercules, California) according to the manufacturer's protocol. Approximately
500 µl BSA-Affiprep was loaded onto empty SPE cartridges (Varian) and
conditioned with PA:SA (3x500 µl). The pH-adjusted samples were
loaded, and the column was washed with PA:SA (3 x 500 µl) followed by
methanol (300 µl). IAA was eluted in five aliquots of methanol, 300 µl
each. The pooled eluate containing free purified IAA was methylated by
incubation with 1 ml ethereal diazomethane for approximately 5 minutes,
evaporated to dryness under N2, and resuspended in 25 µl ethyl
acetate. Quantification was by GC-MS-selected ion monitoring as described by
Ribnicky et al. (Ribnicky et al.,
1996
) using a model 6890N GC/5973 Network MS (Agilent
Technologies, Palo Alto, CA) equipped with an HP-5MS fused silica capillary
column [30 m x 0.25 mm ID, (5%-phenyl)-methylpolysiloxane, 0.25 µm
film thickness (Agilent Technologies)]. Injector temperature was set at
250°C and temperature programmed from 70°C (2 minutes) to 280°C at
20°C min1. Ions at m/z 130, 136, 189 and 195 were
monitored with dwell times of 50 ms per ion.
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Results |
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Roots and shoots of arf6-2 and arf8-3 single mutant and
arf6-2 arf8-3 double mutant seedlings closely resembled those of
wild-type seedlings (data not shown). Other workers found that arf8-1
mutant seedlings had a long hypocotyl
(Tian et al., 2004). However,
we observed a short hypocotyl in dark-grown arf6-2, arf8-3 and
arf6-2 arf8-3 seedlings (Table
1). Primary inflorescence stems of adult arf6-2 and
arf8-3 single mutant plants were 10-20% shorter than those of
wild-type plants (Fig. 2A,
Table 1), whereas numbers of
flowers and of lateral branches were similar to those in wild-type plants
(Table 1; data not shown).
Thus, the short inflorescences of arf6-2 and arf8-3 plants
arose from decreased internode elongation.
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ARF6 and ARF8 are expressed in multiple flower organs
Microarray data indicate that ARF6 and ARF8 are expressed
at multiple floral stages in all flower organs
(Schmid et al., 2005) (see
below). To examine in more detail where ARF6 and ARF8 may be
expressed, we fused 2.1 kb of ARF6 and 2.4 kb of ARF8
promoter sequences to the GUS reporter gene and examined X-gluc
staining in transgenic plants carrying these fusions. Both fusions were
expressed in flowers at multiple stages.
Fig. 4 shows staining patterns
at stages 11, 12 and 13, spanning the period of arrest of double mutant
flowers. For the PARF6::GUS fusion, sepals had staining at
all stages of flower development (Fig.
4A-D). Petals stained strongly at flower stages 9-10 (data not
shown) and this petal staining decreased at stage 11
(Fig. 4A) and disappeared after
flower stage 12. In anthers, staining appeared at stage 11 in the tapetum
(Fig. 4A,H), then disappeared
early in stage 12 when the tapetum degrades
(Sanders et al., 1999
)
(Fig. 4B) and reappeared
throughout the anther late in stage 12
(Fig. 4C). Anther staining
persisted at least until stage 13. In stamen filaments, staining appeared at
stage 11-12, and persisted through stage 13, especially near the apical end of
the filament (Fig. 4B-D). Staining appeared throughout the gynoecium at early stages up to stage 12, and
was especially strong in ovules (Fig.
4A-C). Gynoecium staining weakened somewhat late in stage 12, but
persisted through stage 13, especially near the apical end including the style
(Fig. 4D).
The patterns for PARF8::GUS (Fig. 4E-G,I) were similar to those for PARF6::GUS. PARF8::GUS staining appeared in sepals at all stages, and in petals at stages 9-10. Anthers stained in the tapetum at stage 11 (Fig. 4E,I), and throughout the anther at stages 12-13 (Fig. 4F,G). Filament staining appeared at stage 12 and persisted through stage 13 (Fig. 4F,G). Gynoecia stained throughout at stage 11, and this staining decreased at stages 12-13 (Fig. 4E-G).
These expression patterns correlate with the timing of anther and gynoecium
arrest in arf6-2 arf8-3 double mutant flowers, and suggest that ARF6
and ARF8 are active in anthers and filaments at stage 12 and in gynoecia and
ovules at stages 11 and 12. Nevertheless, additional factors such as the
microRNA miR167, which targets ARF6 and ARF8
transcripts (Allen et al.,
2005; Kasschau et al.,
2003
), or other post-transcriptional effects, may also influence
the timing and location of ARF6 and ARF8 activity in flowers.
Gene expression changes in arf6 arf8 flowers
To identify regulatory changes that occur during flower maturation, we used
Affymetrix microarrays (Redman et al.,
2004) to assess global gene expression patterns in wild-type,
sesquimutant (arf6-2/arf6-2 arf8-3/ARF8) and arf6-2 arf8-3
double mutant flowers. We isolated RNA from pooled flower buds of stages 1-10
(closed buds shorter than 2 mm, also containing a small amount of stem
tissue), stages 11-12 (unopened/opening, corresponding to the stage at which
arf6-2 arf8-3 flowers arrest), or stages 13-14 (flowers open but
organs not yet fallen off), as well as the tops of the inflorescence stem
bearing these flowers (Smyth et al.,
1990
). As arf6-2 arf8-3 double mutant flowers do not
open, for stage 13-14 double mutant flowers we used unopened buds immediately
beneath the youngest stage 11-12 flowers, in the position where stage 13-14
flowers would have been in wild-type inflorescences. In wild-type flowers, of
the 12,300 genes with robust expression levels (see Materials and methods),
1715 differed at least 2.5-fold in expression levels between different floral
stages. Table S1 (in the supplementary material) lists these genes and their
expression levels at each stage in the three genotypes. Three-hundred and
eighty-seven of these genes were expressed at their highest level in stage
1-10 flowers, 417 were expressed at their highest level at stages 11-12, and
911 were expressed at their highest level at stages 13-14
(Fig. 5A). Five-hundred and
ninety-one were also expressed in stems. One-thousand four-hundred and twenty
(83%) of these genes also had stage-specific expression in a recent study that
included floral stages 9, 12 and 15
(Schmid et al., 2005
). These
results indicate that roughly 14% of expressed genes in flowers change in
expression level according to developmental stage.
Gene expression in arf6-2 arf8-3 double mutant flower buds was substantially different. Nine-hundred and forty-two genes were expressed at lower level in double mutant than wild-type flowers at one or more stages, including both 617 developmentally regulated and 325 non-developmentally regulated genes (see Table S2 in the supplementary material, Fig. 5B), and 602 genes were expressed at higher level in double mutant than wild-type flowers at one or more stages (Fig. 5C). Of the 1715 developmentally regulated genes we identified in wild-type flowers, 718 (42%) were no longer differentially regulated at different floral stages in the double mutant (Fig. 5A; see Table S1 in the supplementary material). Moreover, 160 additional stage 13-14 genes were still developmentally regulated in the double mutant before stages 11-12 but no longer increased at stages 13-14 as they did in wild-type flowers. These results indicate that ARF6 and ARF8 control, directly or indirectly, roughly one in 13 genes that are expressed in flowers, and roughly half of developmentally regulated genes in opening flowers. Similar proportions of stage 1-10, stage 11-12, and stage 13-14 genes (defined based on the stage of highest expression in wild-type flowers) were affected (Fig. 5A).
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arf6 arf8 double mutant flowers have decreased auxin response
ARF6 and ARF8 can mediate auxin-induced gene activation
(Ulmasov et al., 1999a). To
determine whether altered auxin response might underlie the developmental
arrest in arf6-2 arf8-3 flower development, we analyzed
global gene expression responses to exogenous auxin in wild-type and mutant
whole floral apices using Affymetrix microarrays (see Materials and methods).
Expression of 35 genes was increased at least twofold in wild-type floral
apices after a 30 minute auxin treatment but changed less than 1.5-fold after
a mock treatment (Table S3 in the supplementary material). Of these 35 genes,
just 23 responded to auxin in the sesquimutant and 14 responded to auxin in
the double mutant. Of the 21 genes that auxin induced in wild-type but not
double mutant floral apices, seven (SAUR62, SAUR63, SAUR64, SAUR65,
SAUR67, SHY2/IAA3 and IAA4) were underexpressed in the double
mutant at one or more stages in the absence of exogenous auxin and were
developmentally regulated at different stages in wild-type flowers (see Tables
S1, S2, S3 in the supplementary material). RNA blot hybridization experiments
that included a 2-hour time point also showed a decrease in auxin induction in
the double mutant treatment with exogenous auxin induced the IAA1,
IAA2, IAA4, IAA5 and IAA16 genes in flowers of wild-type plants,
but induced these genes much less in arf6-2 arf8-3 flowers
(Fig. 5D). These data indicate
that ARF6 and ARF8 are required for maximal auxin response in developing
flowers, and raise the possibility that auxin induces flower maturation. We
also observed that auxin induced 165 genes in double mutant flowers that it
did not induce in wild-type flowers. This finding suggests that flower arrest
may affect auxin response secondarily, and that other ARFs are active in
arf6-2 arf8-3 double mutant flowers.
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We measured JA levels in the same staged flower buds used for the developmental time course microarray experiment. In wild-type flowers, JA levels increased 6.7-fold between stage 1-10 and stage 11-12 buds, and then decreased between stages 11-12 and 13-14 (Table 4). The peak of JA production at stages 11-12, just before anther dehiscence and bud opening, coincides with the timing of the JA requirement revealed by phenotypes of JA-deficient mutants.
The arf6-2 arf8-3 double mutant flowers had a JA level below the
detection limit at all stages (Table
4), indicating that normal JA production in flowers requires ARF6
and ARF8. This activity was specific to flowers, as arf6-2 arf8-3
leaves had wild-type JA levels (data not shown). Sesquimutant flowers, which
do not arrest at stage 12, also had a peak JA level at stages 11-12, but had
lower JA levels than wild-type flowers at all stages. This result suggests
that the level of ARF6 and ARF8 determines the level of JA production,
consistent with the possibility that ARF6 and ARF8 regulate one or more genes
required for JA production. Several JA biosynthetic pathway genes were
underexpressed in double mutant stage 11-12 buds (Table S1 in the
supplementary material), including LOX2 encoding a lipoxygenase
(At3g45140, 1.9-fold higher in wild-type than double mutant stage 11-12 buds),
AOS encoding allene oxide synthase (At5g42650, 2.7-fold higher) and
OPR3 encoding OPDA reductase (At2g06050, 3.2-fold higher). The
promoters of LOX2 and AOS each have two AuxRE motifs within
40 bp of each other and the promoter of OPR3 has two pairs of AuxRE
motifs, suggesting that ARF6 and ARF8 might bind directly to these promoters.
However, although auxin can induce LOX2 and AOS in seedlings
(Tiryaki and Staswick, 2002),
these genes were not induced by auxin in our experiment (see Table S3 in the
supplementary material). Moreover, as JA also induces these genes
(Bell and Mullet, 1993
;
Costa et al., 2000
;
Kubigsteltig et al., 1999
)
these data do not distinguish whether ARF6 and ARF8 induce these genes
directly, or whether the higher JA level in wild-type buds induces them.
Indeed, exogenous JA induced the JA-responsive OPR3 gene normally in
arf6-2 arf8-3 mutant flowers (Fig.
5D), indicating that double mutant flowers could respond to JA,
and suggesting that a low JA level could have caused the decreased
OPR3 expression in the double mutant. The DAD1 gene encoding
a phospholipase required for JA production is expressed in stamen filaments
beginning at stage 12 (Ishiguro et al.,
2001
), suggesting that it could be a target of ARF6 and ARF8.
However, we have failed to find evidence for altered DAD1 level in
arf6-2 arf8-3 flowers (data not shown).
JA feeding experiments revealed that the decrease in JA production contributed to the arf6-2 arf8-3 mutant phenotype. Exogenous JA induced arf6-2 arf8-3 anthers to dehisce (Fig. 2F). When pollen released by anthers of JA-treated arf6-2 arf8-3 flowers was used to fertilize gynoecia of wild-type plants, viable seed were produced, indicating that this JA-rescued pollen was functional. Exogenous JA also increased arf6-2 arf8-3 petal elongation slightly and caused slight but incomplete flower bud opening. By contrast, JA did not rescue the short filaments of arf6-2 arf8-3 stamens, or the developmental defects in carpels. Manual self-pollination of JA-treated arf6-2 arf8-3 double mutant plants produced only a small number of seed. Siliques containing these seed often remained green, and the seed were slow to mature and the embryos sometimes had abnormalities such as fused cotyledons. These results suggest that the decreased JA content of double mutant flowers caused the anther dehiscence defect, but did not cause the stamen filament elongation or gynoecium maturation defects. The JA biosynthetic precursors linolenic acid (18:3) and OPDA also induced anther dehiscence (Fig. 2F), whereas other fatty acids [palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2)] did not. Thus, the regulated step may be upstream of linolenic acid. However, the positive feedback of JA synthesis means that these feeding experiments cannot determine the regulated step unambiguously.
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Discussion |
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Phenotypes of arf6-2 and arf8-3 single mutants,
sesquimutants and arf8-3 gARF6 plants indicate that ARF6 and ARF8
normally act partially redundantly, and that ARF6 and ARF8
gene dosage affects fecundity quantitatively. ARF6 and ARF8 have very similar
DNA-binding and dimerization domains
(Remington et al., 2004;
Ulmasov et al., 1999a
), and
PARF6::GUS and PARF8::GUS have very
similar expression patterns. It therefore seems likely that ARF6 and ARF8 each
regulate common promoters, either as homodimers or heterodimers. They diverge
significantly in the Q-rich middle domains, which are required for gene
activation. Apparently, this divergence does not confer distinct developmental
functions on ARF6 and ARF8, although it might affect the strength of their
activity.
Possible regulatory targets of ARF6 and ARF8
The microarray expression data indicate that arf6-2 and
arf8-3 mutations together have a large effect on gene expression at
all three developmental stages we analyzed. However, the most relevant
regulatory targets may be those that are misexpressed in stage 11-12 buds,
when arf6-2 arf8-3 double mutant flowers arrested. As ARF6 and ARF8
are transcriptional activators, the 472 genes underexpressed at this stage are
more likely to be direct targets. Perhaps the strongest candidates for direct
ARF6 and ARF8 targets are the seven genes that were auxin-induced in wild-type
but not double mutant flowers, developmentally regulated in wild-type flowers,
and underexpressed in double mutant buds (see Table S3 in the supplementary
material). These genes include five SAUR genes from a single clade,
and the sister genes SHY2/IAA3 and IAA4. The functions of
SAUR proteins are unknown. IAA3 and IAA4 may dimerize with ARF6 and ARF8 to
inhibit gene activation activity, and may constitute a negative feedback
loop.
Many genes were expressed at intermediate level in sesquimutant flowers and therefore may be regulated by ARF6 and ARF8 protein concentration. These genes might regulate those phenotypes that ARF6 and ARF8 affect quantitatively, such as stamen development and inflorescence stem elongation. Of the 472 genes that were underexpressed at stages 11-12 in the double mutant, 168 were expressed in the sesquimutant at less than two-thirds (67%) of wild-type level, and are therefore candidates by this criterion. Conversely, genes that were expressed in the sesquimutant at a level close to the level in wild-type flowers are more likely to regulate phenotypes that are not obviously quantitative such as gynoecium maturation, or to have been affected secondarily by the arrest of double mutant flowers at stage 12. These genes might also be regulated preferentially by ARF8 rather than ARF6, and respond to the ARF8 supplied by the single wild-type ARF8 allele present in the sesquimutant.
Phenotypes of arf6-2 arf8-3 flowers and the expression patterns of
ARF6 and ARF8 suggest that ARF6 and ARF8 regulate target
genes in all floral organs. Most of the 942 genes that were underexpressed in
the double mutant were expressed in multiple floral organs in other studies
(Schmid et al., 2005;
Wellmer et al., 2004
),
suggesting that ARF6 and ARF8 regulate a common set of genes in each organ.
However, some of the genes were expressed specifically in particular floral
organs, indicating that ARF6 and ARF8 also have organ-specific effects. For
example, in the Schmid et al. (Schmid et
al., 2005
) dataset, 811 of the 942 genes were expressed in
multiple floral organs, 59 were stamen specific and 13 were carpel specific.
Further work may reveal how ARF6 and ARF8 interact with organ-specific factors
to regulate different genes in different organs. The regulatory targets of
ARF6 and ARF8 that are most relevant to its function in reproduction may be
expressed in stamens and carpels.
JA feeding studies and JA measurements indicate that ARF6 and ARF8 regulate
anther dehiscence by inducing JA production (or decreasing JA conjugation or
breakdown). Several genes encoding JA biosynthetic enzymes are underexpressed
in the double mutant, suggesting that gene expression changes underlie
decreased JA production, although the positive feedback of JA synthesis means
that the gene expression and feeding studies could not reveal the primary step
at which ARF6 and ARF8 regulate JA level. JA in turn presumably regulates
downstream genes required for anthesis. JA is thought to be produced in stamen
filaments (Ishiguro et al.,
2001), and it is therefore possible that ARF6 and
ARF8 expression in filaments stimulates anther dehiscence.
JA did not rescue other arf6-2 arf8-3 mutant phenotypes, and JA-deficient mutants are primarily deficient in anthesis. Therefore, ARF6 and ARF8 must activate other downstream effectors that regulate inflorescence stem elongation, bud opening, filament elongation and carpel maturation. Some genes that depend on ARF6 and ARF8 encode putative regulatory proteins such as transcription factors (Table S4 in the supplementary material), and these may mediate secondary responses to ARF6 and ARF8.
Auxin and flower development
Auxin might activate ARF6 and ARF8 activities by promoting turnover of
Aux/IAA proteins that can inhibit ARF gene induction activity
(Gray et al., 2001;
Tian et al., 2003
;
Tian et al., 2002
;
Tiwari et al., 2003
;
Tiwari et al., 2001
;
Zenser et al., 2001
).
Consistent with this possible mechanism, some auxin-inducible genes were
underexpressed in sesquimutant and double mutant flowers. However, wild-type
flower bud auxin levels did not increase at stages 11-12 or 13-14, suggesting
that a gross increase in auxin level does not induce flower maturation under
our conditions. In fact, auxin is believed to be present in flower primordia
from an early stage and to promote flower bud outgrowth
(Benkova et al., 2003
;
Reinhardt et al., 2003
). Auxin
levels might change more locally, for example, within stamen filaments or
ovules, or auxin may only be limiting under certain growth conditions.
Increased auxin levels have been observed in flowers of a composite species at
the stage of stamen filament elongation
(Koning, 1983
).
Mutations in the ETTIN/ARF3 and MP/ARF5 genes, encoding
two other ARF proteins, affect flower organ numbers, and ettin mutant
flowers also have expanded stigma and style
(Przemeck et al., 1996;
Sessions et al., 1997
). These
patterning defects occur at an earlier stage than the arf6-2 arf8-3
flower arrest at stage 12, and arf6-2 arf8-3 flowers had normal organ
numbers and gynoecium patterning. Different auxin response factors thus
regulate patterning and flower maturation, two very distinct aspects of flower
development. These results imply that different ARF proteins may have some
specificity in the promoters they bind or their interacting partners
(Weijers et al., 2005
).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/18/4107/DC1
* Present address: Center for Integrative Genomics, University of Lausanne,
BEP, 1015-Lausanne, Switzerland
Present address: Department of Genetics, North Carolina State University,
Raleigh, NC 27695-7614, USA
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