• Coordinated regulation of Arabidopsis thaliana development by light and gibberellins

Vol 451 | 24 January 2008 | doi:10.1038/nature06448
LETTERS
Coordinated regulation of Arabidopsis thaliana
development by light and gibberellins
Suhua Feng1,2{, Cristina Martinez1*, Giuliana Gusmaroli1*, Yu Wang3*, Junli Zhou2*, Feng Wang2, Liying Chen2,
Lu Yu2, Juan M. Iglesias-Pedraz4, Stefan Kircher5, Eberhard Schafer5, Xiangdong Fu6, Liu-Min Fan3
¨
& Xing Wang Deng1,2,3
Light and gibberellins (GAs) mediate many essential and partially within the DELLA domain that is required for GA-induced degrada-
overlapping plant developmental processes. DELLA proteins are tion7,8. As expected, TAP–RGAD17 and TAP–GAID17 are completely
GA-signalling repressors that block GA-induced development1. resistant to GA and accumulate at higher levels than wild-type
GA induces degradation of DELLA proteins via the ubiquitin/ proteins, which cannot be further increased by PAC (Fig. 1, and
proteasome pathway2, but light promotes accumulation of
DELLA proteins by reducing GA levels3. It was proposed that – + – + – – MG132
– – + + – – GA3
DELLA proteins restrain plant growth largely through their effect – – – – – + PAC
on gene expression4,5. However, the precise mechanism of their Anti-RGA
function in coordinating GA signalling and gene expression WT
remains unknown. Here we characterize a nuclear protein inter- Anti-RPN6
action cascade mediating transduction of GA signals to the activity
regulation of a light-responsive transcription factor. In the 35S-TAP–
Anti-MYC
absence of GA, nuclear-localized DELLA proteins accumulate to RGL1
Anti-RPN6
higher levels, interact with phytochrome-interacting factor 3
(PIF3, a bHLH-type transcription factor) and prevent PIF3 from Anti-MYC
35S-TAP–
binding to its target gene promoters and regulating gene expres-
RGL2
sion, and therefore abrogate PIF3-mediated light control of Anti-RPN6
hypocotyl elongation. In the presence of GA, GID1 proteins (GA
receptors) elevate their direct interaction with DELLA proteins Anti-MYC
35S-TAP–
in the nucleus, trigger DELLA protein’s ubiquitination and RGL3
Anti-RPN6
proteasome-mediated degradation, and thus release PIF3 from
the negative effect of DELLA proteins. Anti-MYC
35S-TAP–
Light and GA interact during Arabidopsis thaliana seedling RGA
development, regulating hypocotyl elongation, cotyledon opening Anti-RPN6
and light-responsive gene expression; their pathways seem to con-
verge at regulation of the abundance of DELLA proteins (GA path- Anti-MYC
35S-TAP–
way repressors)3,6. Arabidopsis has five DELLA proteins—RGA, GAI, GAI
Anti-RPN6
RGL1, RGL2 and RGL3—defined by their unique DELLA domain
and a conserved GRAS domain4. To analyse them in vivo, we raised Anti-MYC
35S-TAP–
antibodies against endogenous RGA and generated transgenic RGA∆17
Anti-RPN6
Arabidopsis expressing each of the five DELLA proteins with tandem
affinity purification (TAP) tags (Supplementary Fig. 1). The response
Anti-MYC
of DELLA protein levels to exogenously applied GA3 (an active form 35S-TAP–
GAI∆17
of GA) or PAC (paclobutrazol, a GA biosynthesis inhibitor) was Anti-RPN6
examined. We found that one-hour-long GA treatment eliminates
the majority of DELLA proteins, and this GA effect can be largely Figure 1 | Effect of GA3, MG132 and PAC on DELLA protein abundance.
prevented by 100 mM MG132 (a 26S proteasome-specific inhibitor). Immunoblot analysis of RGA (by anti-RGA antibody) and TAP-DELLA
proteins (by anti-MYC antibody) in various light-grown Arabidopsis
PAC, on the other hand, promotes over-accumulation of DELLA seedlings (genotypes labelled to the left of each panel) treated with different
proteins (Fig. 1). These results show for the first time in combinations of GA3, MG132 and PAC. Panels on the left (four lanes) and
Arabidopsis that all the DELLA proteins are under negative control panels on the right (two lanes) are from two independent experiments using
by GA and the proteasome. Next, we generated lines expressing TAP- different protein gel systems. RPN6 immunoblotting (by anti-RPN6
tagged RGAD17 and GAID17, which lack a 17 amino acid motif antibody) is used as a loading control. WT, wild type.
1
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104, USA. 2National Institute of Biological Sciences, Zhongguancun
Life Science Park, Beijing 102206, China. 3Peking–Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, and National Laboratory for Protein Engineering and Plant
Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China. 4Departamento Genetica Molecular de Plantas, Centro Nacional de Biotecnologı
´ ´a-CSIC,
Campus Universidad Autonoma de Madrid, 28049 Madrid, Spain. 5Institut fur Biologie II/Botanik, Albert Ludwigs Universitat, Freiburg D-79104, Germany. 6Institute of Genetics and
´ ¨ ¨
Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China. {Present address: Howard Hughes Medical Institute, University of California at Los Angeles, Los
Angeles, California 90095-1606, USA.
*These authors contributed equally to this work.
475
©2008 Nature Publishing Group
LETTERS NATURE | Vol 451 | 24 January 2008
Supplementary Fig. 1b). Arabidopsis plants that overexpress these promoted by DELLA proteins in the light3,6. We further examined
proteins show a dominant dwarf phenotype, reflecting enhanced the possible mechanism of DELLA proteins in regulating photo-
DELLA activity (Supplementary Fig. 2), which also suggests that morphogenesis. Arabidopsis seedlings have longer hypocotyls on
TAP–DELLA proteins retain normal DELLA function. GA-containing medium, whereas PAC dramatically inhibits the
Inhibition of hypocotyl elongation, an important characteristic of elongation of hypocotyls (Fig. 2a, b). Furthermore, the GA effect is
photomorphogenesis, is shown to be repressed by GA in the dark and more drastic in red light than in dark (Fig. 2b), consistent with the
notion that the endogenous GA level is higher in dark-grown seed-
lings. In addition, 35S-TAP–RGAD17 and 35S-TAP-GAID17 plants
a have much shorter hypocotyls than wild type, which cannot be res-
cued by GA. On the contrary, the hypocotyl of rga-24 gai-t6 double
mutants is longer than that of wild type, and is only partially inhibited
by PAC. In a pentuple mutant (della) of all five DELLA genes, the
hypocotyl length is comparable to that of GA-treated wild type, and
PAC has no significant effect (Fig. 2a, b). Therefore, we reasoned that
WT pif3-1 35S-PIF3 35S-TAP 35S-TAP rga-24 della GA controls hypocotyl growth and affects photomorphogenesis
–HIS–MYC –RGA∆17 –GAI∆17 gai-t6
status, mainly by regulating DELLA protein abundance.
DELLA proteins are proposed to be transcription factors4, and are
b 250 250
Red Red required to localize to the nucleus for their function9,10. Genomic
200 200
studies have revealed a number of GA-responsive genes that are
150 150 regulated by DELLA genes5. However, using the chromatin immuno-
Relative hypocotyl length (%)
100 100 precipitation (ChIP) technique in 35S-TAP–DELLA lines, we were
50 50 unable to observe specific binding of DELLA proteins to any of the
38 GA-responsive gene promoters tested (Supplementary Table 1).
0 0
Thus, we hypothesize that DELLA proteins might regulate gene
140 140
Dark Dark expression indirectly by controlling transcription factors. Because
120 120
100 100
light and DELLA proteins both regulate hypocotyl growth, it seems
80 80 possible that one, or more, of the well-known photomorphogenesis-
60 60 related transcription factors might be a target of DELLA proteins.
40 40 Among them, PIF3 is a good candidate, because it promotes hypo-
20 20 cotyl elongation in red light11—the opposite of DELLA’s function
0
WT 35S-TAP 35S-TAP
0
WT rga-24 della
(Fig. 2a). Moreover, PIF3 has DNA-binding activity12, interacts with
–RGA∆17 –GAI∆17 gai-t6 the active form of phytochrome B (phyB)13,14, and is negatively regu-
lated by phytochrome through the ubiquitin/proteasome path-
c 300 way15–17, indicating it mediates signalling between light and gene
250
expression. We observed that the pif3-1 mutant has a short hypo-
200 cotyl, and is partially resistant to GA and hypersensitive to PAC,
mimicking 35S-TAP–RGAD17 and 35S-TAP–GAID17 plants,
Relative hypocotyl length (%)
150
100 whereas the PIF3 overexpression line shows a long hypocotyl and is
50 hyposensitive to PAC, in a similar manner to GA-treated plants and
0 rga-24 gai-t6 and della mutants (Fig. 2c). These results imply that
GA3 DELLA proteins may negatively regulate PIF3 in the control of hypo-
250
cotyl elongation, representing a convergent point of light and GA
200 pathways (Fig. 2d).
150 This regulation is probably mediated through physical interaction
between PIF3 and DELLA proteins, as suggested by yeast two-hybrid
100
and in vitro pull-down assays (Fig. 3a, d, and Supplementary Fig. 3).
50 Moreover, bimolecular fluorescence complementation (BiFC)
0
analysis detects direct RGA–PIF3 interaction in the nuclei of living
PAC plant cells (Fig. 3b). We further investigated this interaction using an
WT pif3-1 35S-PIF3 35S-TAP 35S-TAP rga-24 della
–HIS–MYC –RGA∆17 –GAI∆17 gai-t6 immunoprecipitation approach. As shown in Fig. 3c, interaction
between RGA and PIF3 is observed in dark-grown seedlings, in which
d PIF3 protein accumulates to reasonable abundance15–17. The inter-
Red light Phytochrome B action is also detectable in red light, when light-induced proteasomal
Hypocotyl elongation
PIF3 by controlling degradation of PIF3 (refs 15–17) is blocked. The interaction is
GA DELLA gene expression dependent on RGA abundance, such that PAC increases RGA–PIF3
GA
receptors proteins
interaction, whereas GA abolishes RGA accumulation and thus PIF3
Figure 2 | DELLA proteins and PIF3 have opposite roles in regulating is released. Importantly, under the condition that RGA–PIF3 inter-
Arabidopsis hypocotyl elongation. a, Images of red-light-grown seedlings. action is enhanced, PIF3’s effect on hypocotyl growth is largely
b, Hypocotyl length measurement (mean 6 s.d.) of untreated seedlings (red), impaired, and vice versa (Figs 2c, 3c), indicating that RGA-bound
or seedlings treated with 10 mM GA3 (blue) or 1 mM PAC (yellow). PIF3 has reduced activity. We tested whether DELLA proteins influ-
c, Hypocotyl length measurement (mean 6 s.d.) of red-light-grown seedlings ence the previously reported interaction between phytochrome and
treated with increasing amounts of GA3 or PAC (see Methods). The
PIF3 (refs 13, 14) by analysing the formation of nuclear speckles
concentrations of GA3 used are 0, 0.5 mM, 1 mM, 2 mM and 5 mM (from left to
right). The concentrations of PAC were 0, 0.01 mM, 0.02 mM, 0.05 mM, 0.1 mM, containing both phyB and PIF3 (ref. 15). Evidently, phyB–PIF3 inter-
0.2 mM and 0.5 mM (from left to right). In b and c, hypocotyl length of action is essentially not affected by altering DELLA protein abun-
untreated wild-type seedlings is set to 100%. d, Simplified diagram depicting dance (Supplementary Fig. 4). Therefore, DELLA protein binding
the genetic interaction of light and GA in the control of hypocotyl elongation most probably affects PIF3’s transcription-regulation activity
by PIF3 and DELLA proteins. della, rga-t2 gai-t6 rgl1-1 rgl2-1 rgl3-1. towards its target genes.
476
©2008 Nature Publishing Group
 NATURE | Vol 451 | 24 January 2008 LETTERS
This notion is supported by the observation that the RGA–PIF3 (LHY; Fig. 3d, and Supplementary Fig. 3), which provides evidence
interaction in vitro is specifically inhibited by pre-incubating PIF3 that RGA–PIF3 and PIF3–DNA bindings are antagonistic. To test
with its cognate binding site, a G-box-containing DNA probe12 this in vivo, we selected five putative PIF3 target genes by analysing
the published literature as well as taking into account results we
a 60 b YFP DAPI obtained from a ChIP microarray analysis focused on PIF3, using a
Bait:
+ PIF3–YFPC
recently reported method18. By ChIP–PCR, we confirmed that these
YFPN–RGA YFPN–RGA
PIF3
β-Gal activity (Miller unit)
50
five promoters are bound by PIF3 as expected. In addition, we found
40 that when DELLA protein level is increased by PAC, PIF3–promoter
30 binding is severely reduced. On the other hand, removing DELLA
+ YFPC
proteins by GA treatment generally leads to enhanced occupancy of
20 PIF3 on the promoters (Fig. 3e). We also noticed that, whereas GA
10
PIF3–YFPC
and PAC do not significantly affect nuclear PIF3–MYC protein levels,
they have slightly opposite effects on PIF3–MYC immunoprecipita-
YFPN +
0 tion (Supplementary Fig. 5), which might be due to higher affinity of
Prey:
or
L1
L2
L3
MYC antibody towards free PIF3–MYC than RGA-bound PIF3–
A
ct
AI
RG
RG
RG
RG
Ve
G
MYC. Among the PIF3 target genes, At5g2120, At4g19030,
c 35S-PIF3–HIS–MYC At2g47890 and At1g34670 show light-responsive expression19.
Interestingly, differential expression of At5g24120, At4g19030 and
Dark Dark Dark Dark Dark Red (1 hour)
At2g47890 have also been reported in genomic studies focused on
gene expression regulation by GA, PIF3 or DELLA genes5,20,21.
l
l
l
l
l
A
A
A
A
ta
ta
ta
ta
ta
e
RG
RG
RG
RG
To
To
To
To
To
Pr
Subsequently, we used PCR with reverse transcription (RT–PCR)
Anti-MYC
to check whether PIF3–promoter binding indeed affects gene
expression. As shown in Fig. 3f, overexpressing PIF3 and reducing
Anti-RGA DELLA protein abundance (della mutant or GA treatment) have
Anti-CAND1 similar effect on the expression of two representative PIF3 target
+ + + – + MG132 genes, whereas increasing DELLA protein abundance (overexpres-
– – – + – GA3 sing RGAD17 or PAC treatment) has the opposite effect. Overall,
– – + – – PAC we demonstrate that DELLA proteins antagonize PIF3 function by
protein–protein interaction and sequestration, which at least partly
d
+ + + + + + + + + + + + HIS–PIF3 explains their effect on gene expression and the coordinated control
+ + + + + + – – – – – – MBP–RGA of hypocotyl growth by light and GA.
– – – – – – + + + + + + MBP
– – – 10 pM 20 pM 40 pM – – – 10 pM 20 pM 40 pM LHY probe We next examined how the GA signal is relayed to affect DELLA
10 pM 20 pM 40 pM – – – 10 pM 20 pM 40 pM – – – G-mut probe protein abundance and thus DELLA–PIF3 interaction. Recently,
Anti-HIS GID1 proteins have been shown to act as nuclear GA receptors22–25.
Input
Through isolating and analysing Arabidopsis gid1 mutants, we
e 35S-PIF3–HIS–MYC Wild type obtained results that are consistent with those reports24,25, suggesting
that GID1s are required for normal GA signalling and participate in
Input MYC IP No antibody MYC IP
light-induced development, possibly by inducing DELLA protein
At5g24120 degradation (Supplementary Note 1 and Supplementary Fig. 6).
At4g19030 We also confirmed the reported GA-dependent GID1–DELLA inter-
At2g47890 action22–25, which requires the 17 amino acid motif within the DELLA
At5g48100 domain, in yeast two-hybrid assays (Fig. 4a, b, and Supplementary
At1g34670 Fig. 7). In addition, BiFC analysis of GID1c and RGA demonstrates
At1g07050 their direct interaction in the nuclei of living plant cells (Fig. 4c). To
– – + – – + – – + – – + GA3 test the effect of GA on GID1–DELLA interaction in planta, we used
+ – – + – – + – – + – – PAC transgenic Arabidopsis expressing each of the three GID1 proteins
with YFP or epitope tags for immunoprecipitation analyses. As
f shown in Fig. 4d, interaction of GID1a with each of the five
A P
G TA
17
∆
–R 5S-
Wild type 35S-PIF3–HIS–MYC DELLA proteins is detectable, and is greatly enhanced by GA.
lla
3
de