1 Department of Biology, University of North Carolina at Chapel Hill, CB #3280,
Coker Hall, Chapel Hill, NC 27599-3280, USA
2 Department of Biochemistry, University of Missouri, Columbia, Columbia, MO
65211, USA
3 Biology Department, Western Washington University, Bellingham, WA 98225-9160,
USA
* Author for correspondence (e-mail: jreed{at}email.unc.edu)
Accepted 27 July 2005
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SUMMARY |
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Key words: Auxin, Auxin response factor, Senescence, Abscission
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Introduction |
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When plant structures have served their purpose, they senesce and are shed
as part of this developmentally programmed sequence of events. Senescence
enables the plant to marshal its resources to maximize its growth and
reproductive capacity, and contributes to pathogen defense and environmental
stress responses. Plant hormones that influence senescence include ethylene,
abscisic acid and jasmonates, which can induce senescence; and auxin,
cytokinin and gibberellins, which can play a role in its suppression
(Lim et al., 2003). Auxin
represses transcription of some genes whose expression is correlated with
senescence and/or abscission (Noh and
Amasino, 1999
; Hong et al.,
2000
; Tucker et al.,
2002
).
The auxin response factor (ARF) family of transcription factors regulate
many responses to auxin. These proteins bind to auxin response elements
(5'-TGTCTC-3') in the promoters of auxin regulated genes and
either activate or repress transcription of these genes
(Ulmasov et al., 1997a).
Arabidopsis has 22 genes encoding ARF proteins
(Remington et al., 2004
). Most
ARFs have three domains: an N-terminal DNA-binding domain, a C-terminal
dimerization domain that is similar to domains III and IV of the Aux/IAA
family of proteins, and a middle region (MR) that activates or represses
transcription (Ulmasov et al.,
1999a
: Ulmasov et al.,
1999b
). ARFs containing glutamine-rich MRs function as activators
of auxin responsive gene expression in transiently transfected protoplasts
(Ulmasov et al., 1999a
) and in
vivo (Wilmoth et al., 2005
;
Okushima et al., 2005a
;
Nagpal et al., 2005
). These
include MP/ARF5, which is involved in embryo patterning and vascular formation
(Hardtke and Berleth, 1998
),
NPH4/ARF7, which is involved in phototropism and gravitropism
(Harper et al., 2000
), ARF19,
which acts redundantly with NPH4/ARF7 in controlling leaf expansion and
lateral root growth (Okushima et al.,
2005a
; Wilmoth et al.,
2005
), and ARF6 and ARF8, which act redundantly in flower
maturation (Nagpal et al.,
2005
). The activity of these ARFs is negatively regulated by
heterodimerization with Aux/IAA proteins
(Reed, 2001
;
Tiwari et al., 2003
).
ARFs containing proline- and/or serine-rich MRs repress auxin responsive
gene expression in protoplast transient assays
(Tiwari et al., 2003). These
include the closely related proteins ARF1 and ARF2. Mutations in ARF2
partially restore apical hook formation to dark-grown hookless1
(hls1) seedlings, and ethylene promotes ARF2 protein turnover in
etiolated seedlings in a HLS1-dependent manner
(Li et al., 2004
). Dark-grown
arf1 arf2 double mutant seedlings had an exaggerated hypocotyl hook,
whereas single mutants resembled wild-type plants, indicating redundancy
between these two genes. These results led to the model that ARF1 and ARF2 act
downstream of HLS1, and that they integrate ethylene and light
signals to control apical hook formation. arf2 mutations restored
asymmetric DR5::GUS expression in apical hooks of dark-grown
hls1 seedlings (Li et al.,
2004
), suggesting that ARF2 may repress auxin-regulated gene
expression. Alternatively the restoration of asymmetric DR5::GUS
expression may have been an indirect effect of restoration of the apical hook.
Other workers have found that arf2 mutant plants have enlarged seeds,
stems and cotyledons, and elongated hypocotyls under red light, but did not
find significant effects on auxin-responsive gene expression (Schruff et al.,
2005; Okushima et al.,
2005b
).
In this report, we show that ARF2 and ARF1 promote transitions between developmental phases. Lines carrying T-DNA insertions in these genes were isolated and lines with decreased expression of ARF2 were also created using double-stranded RNA interference (dsRNAi). The plants with decreased ARF2 flowered late, and had delayed senescence of rosette leaves and delayed abscission of floral organs. In addition ARF1 acted as a repressor of auxin-induced genes. arf1 and arf2 mutations had synergistic effects on some phenotypes and independent effects on others, indicating both redundancy and specialization of ARF1 and ARF2 function. Furthermore, nph4/arf7 and arf19 mutations also enhanced the arf2 senescence and abscission phenotypes, suggesting that ARFs of different functional classes can regulate common processes.
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Materials and methods |
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Analysis of ARF and AUX/IAA gene expression
PARF1::GUS reporter lines were created by digesting DNA
from BAC AC007258 with restriction endonucleases XbaI and
SacI. A 2.6 kb fragment encompassing the ARF1 promoter and start
codon was cloned into pBluescript SK+ (Promega). This plasmid was used as a
template to amplify a 2.2 kb fragment containing the ARF1 5' upstream
regions using the T7 primer and the gene specific primer
5'-GGAAGCTGCCATGGGGAATC-3'. This fragment was cloned into the
SalI/NcoI sites of the pEBGUS vector
(Hagen et al., 1991). The ARF1
promoter-GUS-nos fragment was excised with EcoRI and cloned into
pPZP211. PARF2::GUS lines were created by amplifying a 2.5
kb fragment from BAC AB016880 DNA using the primers:
5'-CACACAAATGCTGCAGAGTACTCTTGGGTC-3' and
5'-CGCAGGATCCGAAGCTCAGATCTGTTTCATCTGG-3'. Seven plant lines per
construct were analyzed for GUS activity and representative lines were chosen
for further analysis. PIAA3::GUS and
PIAA7::GUS lines were described previously
(Tian et al., 2002
). Plant
tissue was stained for GUS activity as described previously
(Wilmoth et al., 2005
).
Total RNA was isolated from seedlings grown in liquid medium as described
previously (Tian et al., 2002)
or from leaves or flowers of adult plants using Trizol reagent (Invitrogen
Life Technologies, Carlsbad, CA). Poly (A+) RNA was extracted using
oligo(dT)25 Dynabeads according to manufacturers' instructions
(Dynal, Lake Success, NY). RNA gel blot hybridizations were performed as
described (Nagpal et al., 2000; Tian et
al., 2002
) using 30 µg of total RNA or mRNA derived from 50
µg of total RNA. Probes were created by PCR using genomic DNA or cDNA as
template and the following primers: ARF1,
5'-CCTAATGGCAGCTTCCAATCAT-3' and
5'-GCAAATGACGCCTTGGTTGGC-3'; ARF2,
5'-GAAGGTATGGCGAGTTCGGAGG-3' and
5'-GGAGGCTGTCGAGACATATC-3'; IAA3,
5'-CTTCATCATCAGCAGCTTCTCT-3' and
5'-GGGGATGTTTCAGTTTTCCTT-3'; IAA7,
5'-CAAGTAACATGATCGGCCAAC-3' and
5'-GGCTTAGAAGGATCTTTAAGGGGTA-3'; SAG12,
5'-GATGTTGTTGGGCGTTTTCAGCGG-3' and
5'-CTTTCATGGCAAGACCACATAGTCC-3'; IAA2,
5'-CTTATGATCCAGAGAAGCTGAGAATC-3' and
5'-ATCTCATGTATCTTTACATCAAACTTC-3', and IAA5,
5'-TTCCCATGAGAACATATAAAGTGG-3' and
5'-TGACTCTTTTTCGCCGGTTC-3'; PDF1.2,
5'-ATGGCTAAGTTTGCTTCCATCATC-3' and
5'-ACATGGGACGTAACAGATACACTT; ERF1,
5'-CCACTTCAAACTTAAGGTCCCTA-3' and
5'-ATGGATCCATTTTTAATTCAGTCC-3'; ARF7,
5'-TCCTGCTGAGTTTGTGGTTCCTT-3' and
5'-GGGGCTTGCTGATTCTGTTTGTTA-3'; and ARF19
5'-ACGATTGCTGTTGCTAACCA-3' and
5'-GGAATGCTGGGAATACCAAA-3'.
Determination of chlorophyll levels
For senescence assays, plants were grown under short day conditions for 6
weeks, at which time the plants had approximately 18 leaves visible to the
naked eye. The 9th and 10th leaves were excised and either frozen in liquid
nitrogen for RNA extraction or placed in a microfuge tube containing 50 µl
of water so that the petiole was immersed in water. The microfuge tubes were
then placed in the dark for various lengths of time. Chlorophyll content was
determined spectrophotometrically and normalized to fresh weight as described
previously (Porra et al.,
1989).
Histology
Samples were fixed overnight in 4% glutaraldehyde in 50 mM potassium
phosphate buffer, pH 7.0, then washed twice with buffer. Samples were
dehydrated in a graded ethanol series to 100% ethanol, then embedded in London
Resin White medium grade resin (Sigma) according to manufacturer's
instructions. Sections (1 µm) were obtained using a Sorvall Porter-Blum
MT2-B ultramicrotome, heat-fixed to a glass slide, then stained with 0.5%
Toluidine Blue.
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Results |
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ARF2 mRNA was not detected in the arf2-8 line (Fig. 1B), which contains an insertion in the middle region. We also made a dsRNAi construct to silence the ARF2 gene (Fig. 1A) and transformed this construct into Columbia, Ws-0 and arf1-4 backgrounds. In the Columbia background, 32 out of 44 T1 plants exhibited decreased fertility and delayed floral organ abscission similar to arf2-8 (see below). In the Ws-0 and arf1-4 backgrounds, two out of eight and 37 out of 58 T1 plants, respectively, displayed wild-type fertility but exhibited delays in floral organ abscission. Five arf1-4 dsARF2 T1 plants exhibited decreased fertility, similar to arf2-8 plants. These plants also had delayed floral organ abscission. The transgene from a strong arf1-4 dsARF2 line that segregated 3:1 for antibiotic resistance was crossed into the Ws-0 background (see Materials and methods) and this line was further characterized in this study. A reduced amount of ARF2 transcript was observed in this dsRNAi line (Fig. 1B), indicating that these plants may retain some ARF2 function.
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We also used a detached leaf assay to study the effects of arf1
and arf2 mutations on dark-induced senescence in plants prior to
flowering. Although some differences in gene expression patterns exist between
natural and dark-induced senescence (Becker
and Apel, 1993; Weaver et al.,
1998
), detached leaf assays have been used as a convenient method
of accelerating leaf senescence (Oh et
al., 1997
; Lin and Wu, 2004). Rosette leaves from 6-week-old
plants that had been grown under short day conditions were placed in darkness
for 8 days prior to chlorophyll measurement. Under these conditions,
arf1-4 and arf1-5 leaves lost a similar amount of
chlorophyll, as did leaves of wild-type plants, whereas arf2-8, Ws-0
dsARF2, arf1-5 arf2-8 and arf1-4 dsARF2 plants retained more
chlorophyll than did wild-type plants (Fig.
3A). Most of the chlorophyll retention was attributed to the loss
of ARF2 and a time course was conducted to compare Columbia and
arf2-8 plants. Columbia leaves lost chlorophyll at a faster rate than
did arf2-8 leaves, starting after 2 days in darkness
(Fig. 3B).
To determine whether other aspects of leaf senescence were affected, RNA
gel blots were used to examine the expression of SENESCENCE ASSOCIATED
GENE 12 (SAG12), a marker for senescence
(Lohman et al., 1994). Whereas
many SAG genes respond to multiple senescence-inducing factors
(He et al., 2001
),
SAG12 is specifically activated by developmental regulation and not
by hormone- or stress-controlled pathways
(Weaver et al., 1998
) and is
therefore an excellent marker gene for development-mediated senescence. Leaves
from Columbia plants that had been placed in the dark accumulated
SAG12 mRNA starting after 2 days, and SAG12 levels peaked
after 4 days (Fig. 3C).
arf2-8 leaves accumulated very little SAG12 even after 6
days (Fig. 3C). These data show
that ARF2 is required for normal leaf senescence both in intact plants and in
detached leaves. By contrast, SAG12 expression levels and chlorophyll
loss were similar in Ws-0 and arf1-4 plants, suggesting that
ARF1 on its own has little effect on senescence.
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Younger, expanding leaves of PARF2::GUS::ARF2 plants also stained in the vasculature and ground tissue, both in freshly detached leaves and in those placed in the dark for 4 days (Fig. 4E,F), suggesting that ARF2 may also function early in leaf development during leaf expansion.
In contrast to ARF2, ARF1 mRNA decreased as senescence progressed in both Columbia and arf2-8 plants (Fig. 3C). ARF1 mRNA levels were quantified in three independent blots and were found to be slightly higher (2.7±0.9-fold) in freshly detached arf2 leaves when compared with Columbia leaves. These patterns suggest that ARF1 has a different function than does ARF2 during wild-type leaf senescence.
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In Arabidopsis, shortly after flowers open and pollination occurs,
fertilized siliques begin to expand, and sepals, petals and stamens wither and
abscise (Smyth et al., 1990).
We measured timing of organ abscission in two ways. The first method was to
identify the position of the first flower whose perianth organs had been shed,
where newly opened flowers are designated as position 1 and flowers are
numbered basipetally onwards (Patterson,
2001
; Patterson and Bleecker,
2004
). Wild-type and arf1 single mutant plants typically
shed perianth organs at approximately position 6.5, whereas Ws-0
dsARF2 and arf2-8 plants typically retained their perianth
organs until about position 8 (Table
1). The arf1-4 dsARF2 plants and arf1-5 arf2-8
lines did not shed floral organs until positions 9.6 and 9.0, respectively.
The second method of determining the timing of organ abscission was to measure
the length of the silique at abscission. In Ws-0 and Columbia, flowers shed
their perianth organs when the silique was
4.5 mm long
(Table 1), with fully expanded
siliques reaching a length of 11.3±1.0 mm. arf1-4 and
arf1-5 plants did not differ significantly from wild-type plants.
However, in arf2-8 and dsARF2 plants, floral organ
abscission was delayed until the siliques reached
10.0 and 9.0 mm long,
respectively. The delay of organ shedding was also more pronounced in the
arf1-5 arf2-8 plants and in arf1-4 dsARF2 lines, which did
not shed perianth organs until the silique was 10.6 and 10.2 mm, respectively
(Fig. 5F,G). In addition to
being shed later than wild-type sepals, sepals of arf2 and arf1
arf2 plants remained green and turgid until detachment, whereas wild-type
sepals turned yellow prior to detachment
(Fig. 5B,C).
In these experiments, arf2 single and double mutant lines were manually pollinated to ensure that differences in perianth shedding were not due to changes in signals from fertilized siliques to outer whorl organs. However, manual pollination did not affect the position at which floral organs were shed (data not shown). Furthermore, whereas strong arf1-4 dsARF2 lines were infertile, weaker lines set seed normally. These fertile lines also exhibited delays in floral organ abscission (Fig. 5G), confirming that delays in floral organ abscission did not correlate with sterility.
Cells destined to become the abscission zone can be identified prior to
separation by their morphology. Abscission zone cells are located at the base
of floral organs and are typically smaller and more densely cytoplasmic than
neighboring non-separating cells (Roberts
et al., 2002). To determine whether abscission zones formed
normally in the mutant, flowers from position 4 (two flowers prior to floral
organ abscission in wild-type plants) were sectioned from Columbia and
arf1-5 arf2-8 plants (Fig.
5D,H). In both plants, an abscission zone comprising a few layers
of small cells was present near the base of the floral organs. Hence, at this
level of resolution, the abscission zones looked similar in both mutant and
Columbia plants. This suggests that the formation of the abscission zone
proceeded normally in arf2 plants, but the timing of cell separation
was delayed. This result is consistent with those of Hanisch Ten Cate and
Bruinsma (Hanisch Ten Cate and Bruinsma,
1973
) who found that although IAA treatment delayed abscission in
Begonia flower bud pedicels, it did not affect the anatomy of the
abscission zone. Auxin inhibits the expression of some cell wall degrading
enzymes (Hong et al., 2000
;
Tucker et al., 2002
) and may
therefore delay cell separation rather than abscission zone patterning.
Two to three weeks after pollination, Arabidopsis siliques dry and shatter, releasing the mature seed. ARF1 and ARF2 also influenced the timing of silique dehiscence. Dehiscence was delayed by approximately 4 days relative to pollination in arf2 and arf1 arf2 mutants (Table 1). We also observed that siliques of arf2 plants remained green for longer than wild-type siliques.
In summary, arf2 mutations had a major effect on senescence of rosette leaves, stamen length, floral organ abscission, flowering time and silique dehiscence. Mutations in the ARF1 gene typically enhanced the effect of arf2 mutations. This was evident in both the arf1-4 and arf1-5 alleles for stamen elongation and the timing of floral organ abscission. However, only the stronger, transcript null arf1-4 allele also enhanced the delayed leaf senescence and flowering time phenotypes.
Expression of ARF1 and ARF2
ARF1 and ARF2 mRNA are each present in roots, rosette
leaves, cauline leaves and flowers
(Ulmasov et al., 1999b). To
examine the expression domains of ARF1 and ARF2 further, we
made transgenic plant lines carrying 2.2 kb of the ARF1 promoter or
2.5 kb of the ARF2 promoter upstream of the GUS gene and
examined GUS expression patterns by X-Gluc staining. In both
PARF1::GUS and PARF2::GUS lines,
X-Gluc staining appeared throughout 8-day-old seedlings and in rosette leaves
(data not shown). In addition, stain appeared in the sepals and carpels of
young flower buds of both PARF1::GUS and
PARF2::GUS lines (Fig.
6A,D). Staining in the carpels became restricted to the style at
approximately stage 10, at which time staining also appeared in anthers and
filaments. Sepal, stamen and carpel staining persisted until floral organs
were shed (Fig. 6B,E). From
stage 13, GUS activity also appeared in the region at the top of the pedicel,
including the abscission zone (Fig.
6C,F). PARF2::GUS::ARF2 plants
(Li et al., 2004
) showed
similar staining patterns as PARF2::GUS plants (data not
shown), suggesting that the PARF2::GUS patterns observed
reflect the true expression pattern of ARF2. Furthermore, the GUS activity was
consistent with microarray expression profiles that found ARF1 and
ARF2 mRNA in all four floral whorls
(Schmid et al., 2005
).
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Whereas arf1-4 plants had strongly derepressed PIAA3::GUS and PIAA7::GUS activity, increased GUS activity was detected in some but not all arf1-5 plants carrying PIAA3::GUS or PIAA7::GUS fusions. This may reflect differences in the nature of the insertions, as the arf1-4 allele is a transcript null mutant and probably stronger than the arf1-5 allele, which produces some ARF1 transcript. Alternatively, the difference between the two alleles may be due to differential effects between the Ws-0 and Columbia ecotypes.
Although arf2 mutants had stronger organ abscission phenotypes than arf1 mutants, no stain was visible in the floral abscission zones of arf2-6 or Ws-0 dsARF2 plants carrying PIAA3::GUS or PIAA7::GUS fusions (data not shown). arf1-4 arf2-8 lines stained in the same locations as did arf1-4 lines (Fig. 6K,L,Q,R). It is possible that ARF2 also represses IAA gene expression, but the upregulation of ARF1 in the arf2 background (Fig. 3C) may counteract the loss of ARF2 and result in the overall lack of ectopic expression of PIAA3::GUS and PIAA7::GUS in arf2-8 plants. However, PIAA3::GUS and PIAA7::GUS stain appeared similar in both the arf1-4 and arf1-4 arf2-8 flowers, and they had similar levels of IAA3 and IAA7 mRNA, indicating that ARF2 had no further effect on the expression of these genes.
Taken together, these results indicate that ARF1 and ARF2 have distinct
effects on gene expression in flowers. ARF1 represses expression of some
auxin-inducible genes, consistent with its ability to repress gene expression
in protoplast assays (Tiwari et al.,
2003; Ulmasov et al.,
1999b
). However, mutation of ARF2 did not affect
expression of these genes.
ARF2 functions independently of ethylene and cytokinin response pathways
Ethylene insensitive plants such as etr1 and ein2 have
some characteristics in common with arf2 plants, such as delayed
floral organ abscission and delayed leaf senescence
(Bleecker and Patterson, 1997).
We therefore made arf2-8 ein2-1 double mutant lines to determine how
ARF2 might interact with the ethylene signaling pathway to promote
senescence. Both ein2 and arf2 single mutant leaves had
reduced dark-induced chlorophyll loss. After incubation in the dark for 8
days, Columbia leaves retained only 18% of their chlorophyll, while
arf2-8 and ein2-1 leaves retained 45% and 49%, respectively
(Fig. 7A). arf2-8
ein2-1 leaves retained 76% of their chlorophyll, indicating that the
mutations act additively and that delayed senescence in arf2 plants
cannot be attributed solely to alterations in ethylene signaling.
arf2 and ein2 mutations also had an additive effect on the number of opened flowers that retained their floral organs. Whereas Columbia flowers shed their perianth at position 5.3, arf2-8 and ein2-1 flowers shed their perianth at positions 8.8 and 7.3, respectively, and flowers from arf2-8 ein2-1 shed their outer whorl organs at position 10.2 (Fig. 7B).
Ethylene induces expression of PDF1.2 and ERF1 in rosette
leaves (Penninckx et al.,
1998; Solano et al., 2000). Exogenous ethylene induced these genes
to a similar level in both Columbia and arf2-8 leaves, suggesting
that arf2-8 plants have normal sensitivity to exogenous ethylene
(Fig. 7C). Furthermore,
detached arf2-8 rosette leaves produced similar amounts of ethylene
as Columbia leaves (data not shown). These results indicate that ARF2 controls
senescence and floral organ abscission independently of ethylene.
PSAG12::IPT plants that produce a burst of cytokinin in
response to the onset of senescence show delays in leaf senescence
(Gan and Amasino, 1995) and
disruption of the cytokinin signaling pathway affects the rate of dark-induced
chlorophyll loss (To et al.,
2004
). Auxin and cytokinin are known to interact on many levels
(Coenen and Lomax, 1997
) and it
is therefore possible that the effect of ARF2 on senescence is mediated by
cytokinin. To test this hypothesis, detached leaves from Columbia and
arf2-8 plants were treated with cytokinin to determine whether the
mutant plants respond differentially to hormone treatment
(Fig. 7D). Cytokinin delayed
chlorophyll loss in both wild-type and mutant leaves. Although arf2-8
leaves senesced more slowly than Columbia leaves, the relative delay in
chlorophyll loss caused by exogenous cytokinin appeared similar for both
genotypes. It therefore seems unlikely that delayed senescence of
arf2-8 leaves was due to alterations in cytokinin sensitivity.
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In addition, the anthers of arf2-8 nph4-1 arf19-4 flowers did not
dehisce and the plants were therefore male sterile. Unlike arf6 arf8
flowers (Nagpal et al., 2005),
jasmonic acid did not rescue the anther dehiscence phenotype. Whereas
nph4 and arf19 enhanced the delayed senescence of
arf2, the arf2 mutation did not affect the leaf expansion or
gravitropism phenotypes of nph4 arf19 plants.
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Discussion |
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Does ARF2 promote these phenotypes by a common mechanism? The abscission of floral organs and silique dehiscence phenotypes involve delays in senescence and may therefore be regulated by similar mechanisms as rosette leaf senescence. More generally, ARF2 may control production of some metabolic signal which in turn regulates these transitions as well as flowering and stamen development. Leaf and lateral root initiation and the timing of abaxial trichome production proceeded normally in arf2 plants (data not shown), so the developmental delays seen are not likely due to slower overall growth or to slower phase change. Thus, if ARF2 controls these transitions via a common signal, it is unlikely to be a growth-limiting substance.
An alternative model is that ARF2 acts through distinct mechanisms in
different tissues and/or at different developmental stages. Consistent with
this idea, ARF2 is a target of ethylene signaling in etiolated seedlings
(Li et al., 2004), but appears
to act independently of ethylene in senescing leaves and flowers. In addition,
arf2 mutants have abnormally large inflorescence stems and ovule
integuments caused by increased cell numbers, suggesting that ARF2 may
normally inhibit growth and cell division in ovules and stems (Schruff et al.,
2005; Okushima et al., 2005b
).
However, these phenotypes might have arisen if arf2 mutations prolong
the period of stem and ovule growth, thereby allowing time for extra cell
divisions to occur. In other tissues, increasing the duration of a `growth'
period might in effect delay the transition to a subsequent stage, as we
suggest occurs in leaves and flowers of arf2 mutants.
ARF1 and ARF2 have both distinct and overlapping functions
Of the 22 ARF genes in Arabidopsis thaliana, ARF2 is most similar
to ARF1 (Remington et al.,
2004). However, arf1 plants had no defects in senescence,
flowering time, abscission of floral organs, fertility, auxin-mediated lateral
root initiation, auxin-inhibited root elongation, hypocotyl elongation in
response to different light regimes, gravitropism, phototropism or shoot
branching (data not shown). Although arf1 mutations did not confer
phenotypes on their own, they did enhance late flowering, floral organ
abscission and stamen elongation phenotypes of arf2-8 and Ws-0
dsARF2 plants, and the delayed leaf senescence of Ws-0
dsARF2 plants. Similarly, arf1 mutations enhanced the
effects of arf2 mutations on apical hook formation
(Li et al., 2004
). These
results indicate that ARF1 and ARF2 have some functions in common.
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The ethylene precursor ACC increased expression of the auxin-responsive
reporter gene DR5::GUS (Ulmasov
et al., 1997b) in etiolated arf2 seedlings more than in
wild-type plants (Li et al.,
2004
), showing that increased expression of auxin-regulated genes
in arf2 plants may occur in response to certain stimuli. However, we
and others have failed to detect consistent alterations in auxin-induced gene
expression in arf2 mutants (Fig.
6S) (Okushima et al.,
2005b
) (M. C. Schruff, M. Spielman and R. J. Scott, personal
communication), suggesting that ARF2 does not suppress auxin-induced gene
expression in a general manner. It therefore seems likely that ARF2 function
involves additional mechanisms, and ARF2 may not conform to the canonical
auxin response model. Phylogenetic studies indicate that ARF1 and
ARF2 diverged prior to the monocotdicot split
(Remington et al., 2004
;
Sato et al., 2001
) and thus
may have had ample time to evolve distinct biochemical activities.
Activating ARFs also affect senescence and abscission
Mutations in NPH4/ARF7 and ARF19 did not affect
senescence on their own, but enhanced arf2-8 phenotypes as did
mutations in ARF1. Unlike ARF1, which represses gene expression,
NPH4/ARF7 and ARF19 activate gene expression in protoplast assays and in vivo
(Okushima et al., 2005a;
Wilmoth et al., 2005
).
Expression of ARF2, NPH4/ARF7 and ARF19 increased in
response to senescence, and promoter::GUS fusions to all three (as well as the
PARF2::GUS:ARF2 protein fusion) were expressed in leaf vasculature
(Okushima et al., 2005a
;
Wilmoth et al., 2005
). In
addition, ARF1, ARF2, NPH4/ARF7 and ARF19 are all expressed
at the base of the flower, including the abscission zone (this study)
(Wilmoth et al., 2005
). These
results suggest that ARF1, ARF2, NPH4/ARF7 and ARF19 are all present in the
same tissues and might interact together in the same cells. MADS-box proteins
may regulate floral organ identity in higher-order complexes
(Honma and Goto, 2001
), and
ARFs may interact analogously. For example, ARF2 might recruit activating ARFs
to promoters that they would otherwise recognize poorly. In protoplast assays,
the ability of ARFs to regulate promoter activity depended on the arrangement
of AuxREs, suggesting that ARFs may indeed have different specificities for
different promoters (Tiwari et al.,
2003
). Alternatively, different ARFs may target different
promoters and thus affect different aspects of senescence. Aux/IAA proteins
can inhibit transcriptional activation by activating ARFs
(Tiwari et al., 2003
;
Tiwari et al., 2004
),
potentially adding further regulatory inputs.
Auxin and senescence
Classical studies have correlated auxin levels with senescence and
abscission (reviewed in Addicott,
1982; Nooden and Leopold,
1988
; Sexton and Roberts,
1982
). In bean leaves, a gradient of auxin levels was detected
between the leaf blade and the stalk. Auxin levels declined with leaf age and
senescence occurred when auxin levels between the leaf and stalk were
approximately equal (Shoji et al.,
1951
). Application of IAA to the distal end of abscission zone
explants delayed abscission, while addition to the proximal end promoted
abscission (Addicott and Lynch,
1951
). This suggests that changes in auxin gradients may signal
the onset of or enhance senescence and may explain the observation that
treating whole plants with auxin had little effect on abscission
(Addicott et al., 1955
). These
conclusions also raise the possibility that ARF1, ARF2, NPH4/ARF7 and ARF19
may interact in some way to read auxin gradients.
Comparisons to other delayed senescence and abscission mutants of Arabidopsis
Transgenic plants that overexpress the MADS transcription factor gene
AGL15 also had delayed flowering, floral organ abscission and fruit
ripening (Fernandez et al.,
2000; Fang and Fernandez,
2002
). Those authors speculated that AGL15 may be involved in
maintaining plants in a juvenile state. As ARF2 accelerates the same
transitions, ARF2 may therefore antagonize AGL15 to control the developmental
progression of plant aging. However, arf2 plants show some
abnormalities that 35S::AGL15 plants do not, such as delayed leaf
senescence and reduced fertility. arf2 mutants had normal
AGL15 expression levels, and the PARF2:GUS fusion
was expressed normally in 35S::AGL15 plants (data not shown),
suggesting that ARF2 and AGL15 do not regulate the
expression of one another.
A number of other Arabidopsis mutant plants also have delays in
floral organ abscission (Jinn et al.,
2000; Patterson and Bleecker,
2004
; Butenko et al.,
2003
; Fernandez et al.,
2000
), but differ from arf2 plants in significant
respects. Whereas abscission is delayed by three or four flowers in
arf2 plants, the inflorescence deficient in abscission
(ida) mutant has a complete loss of floral organ shedding
(Butenko et al., 2003
). The
ida gene is predicted to encode a 77 amino acid peptide that may act
as a ligand for a protein such as HAESA, a receptor-like protein kinase whose
antisense suppression resulted in a delay of floral organ abscission
(Jinn et al., 2000
). In a
screen for delayed floral organ abscission (dab) mutants, three loci
were identified (Patterson and Bleecker,
2004
). Sepals of dab2 and dab3 plants turned
yellow prior to detachment, but dab1 sepals, like arf2
sepals, remained green and turgid. However, all other aspects of plant growth
appeared normal in these plants, whereas arf2 plants had multiple
defects throughout plant development.
Arabidopsis mutants displaying delays in leaf senescence (for a
review, see Lim et al., 2003)
include plants with mutations in hormone signaling pathways such as ethylene
(Grbic and Bleecker, 1995
) and
cytokinin (To et al., 2004
).
arf2-8 plants were as sensitive to exogenous cytokinin as wild-type
plants, implying that ARF2 affects senescence independently of cytokinin. The
ethylene response mutant ein2 has a phenotype similar to that of
arf2 in both leaf senescence and delayed floral organ abscission.
Although ethylene and ARF2 each promoted senescence, they have
opposing effects on apical hook formation, and in etiolated seedlings ethylene
promotes the degradation of ARF2 protein
(Li et al., 2004
). However, in
mature plants, ARF2 appears to function independently of the ethylene
signaling pathway as arf2 and ein2 mutations had additive
effects on leaf senescence and floral organ abscission. Ethylene levels
increase in senescing leaves, yet ARF2 transcript and GUS::ARF2
fusion protein increased in detached leaves undergoing dark-induced
senescence. It is therefore unlikely that ethylene causes the depletion of
ARF2 in mature leaves. Taken together, these results suggest that, unlike
during apical hook formation, ARF2 does not act downstream of ethylene in the
control of leaf senescence or floral organ abscission.
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ACKNOWLEDGMENTS |
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