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Light activates the degradation of PIL5 protein to promoteseed germination through gibberellin in Arabidopsis

Eunkyoo Oh1, Shinjiro Yamaguchi2, Yuji Kamiya2, Gabyong Bae1, Won-Il Chung1 and Giltsu Choi1,*

1Department of Biological Sciences, KAIST, Daejeon 305-701, Korea, and2RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan

Received 25 January 2006; revised 14 March 2006; accepted 21 March 2006.*For correspondence (fax þ82 42 869 2610; e-mail [email protected]).

Summary

Angiosperm seeds integrate various environmental signals, such as water availability and light conditions, to

make a proper decision to germinate. Once the optimal conditions are sensed, gibberellin (GA) is synthesized,

triggering germination. Among environmental signals, light conditions are perceived by phytochromes.

However, it is not well understood how phytochromes regulate GA biosynthesis. Here we investigated

whether phytochromes regulate GA biosynthesis through PIL5, a phytochrome-interacting bHLH protein, in

Arabidopsis. We found that pil5 seed germination was inhibited by paclobutrazol, the ga1 mutation was

epistatic to the pil5 mutation, and the inhibitory effect of PIL5 overexpression on seed germination could be

rescued by exogenous GA, collectively indicating that PIL5 regulates seed germination negatively through GA.

Expression analysis revealed that PIL5 repressed the expression of GA biosynthetic genes (GA3ox1 and

GA3ox2), and activated the expression of a GA catabolic gene (GA2ox) in both PHYA- and PHYB-dependent

germination assays. Consistent with these gene-expression patterns, the amount of bioactive GA was higher

in the pil5 mutant and lower in the PIL5 overexpression line. Lastly, we showed that red and far-red light

signals trigger PIL5 protein degradation through the 26S proteasome, thus releasing the inhibition of bioactive

GA biosynthesis by PIL5. Taken together, our data indicate that phytochromes promote seed germination by

degrading PIL5, which leads to increased GA biosynthesis and decreased GA degradation.

Keywords: PIL5, phytochrome, seed germination, gibberellin, protein degradation, Arabidopsis.

Introduction

As photosynthetic organisms, plants must carefully monitor

external light conditions and adjust their growth and

development accordingly. In angiosperms, at least three

photoreceptor systems – phototropins (PHOTs); crypto-

chromes (CRYs); and phytochromes (PHYs) – are respon-

sible for monitoring light conditions and responding by

making adjustments to various physiological and develop-

mental processes (Fankhauser and Staiger, 2002). Among

these, phytochromes perceive red and far-red lights and

regulate various processes, including seed germination,

seedling development, chloroplast development, shade

avoidance and flowering (Chory et al., 1996; Neff et al., 2000;

Sullivan and Deng, 2003).

In Arabidopsis, phytochromes consist of five members

that can be grouped into the type I phytochrome (PHYA),

which functions as a far-red light receptor; and the type II

phytochromes (PHYB, PHYC, PHYD, and PHYE), which

function as red light receptors (Quail, 1998). Regardless of

their spectral specificities, both types of phytochrome

regulate similar physiological processes by triggering tran-

scriptional cascades that ultimately alter the expression of

10–30% of the entire transcriptome (Ma et al., 2001; Tepper-

man et al., 2001). Various transcription factors are known to

be centrally involved in the transcriptional cascades of light

signalling, including COG1 and OBP3 (encoding DOF family

members); HY5 and HYH (encoding bZIP family members);

LAF1 (encoding a MYB family member); and HFR1, PIL1,

PIL5, PIL6, PIF3 and PIF4 (encoding bHLH family members)

(Ballesteros et al., 2001; Chattopadhyay et al., 1998; Fairchild

et al., 2000; Fujimori et al., 2004; Holm et al., 2002; Huq and

Quail, 2002; Huq et al., 2004; Kim et al., 2003; Monte et al.,

2004; Ni et al., 1998; Oh et al., 2004; Park et al., 2003; Salter

et al., 2003; Ward et al., 2005). Phytochromes initiate tran-

scriptional cascades by modulating the activities of these

124 ª 2006 The AuthorsJournal compilation ª 2006 Blackwell Publishing Ltd

The Plant Journal (2006) 47, 124–139 doi: 10.1111/j.1365-313X.2006.02773.x

transcription factors at the transcriptional or post-transla-

tional levels (Bauer et al., 2004; Duek et al., 2004; Fairchild

et al., 2000; Holm et al., 2002; Huq and Quail, 2002;

Jang et al., 2005; Oyama et al., 1997; Park et al., 2003,

2004; Salter et al., 2003; Seo et al., 2003; Yamashino et al.,

2003; Yang et al., 2005).

Different signalling components regulate different light

responses. The best studied of these is the inhibition of

hypocotyl elongation by phytochromes, in which a large

proportion of the light signalling components interact in

varying manners. Some signalling components regulate

both PHYA- and PHYB-mediated inhibition of hypocotyl

elongation (e.g. HY5); some regulate only PHYB-mediated

inhibition of hypocotyl elongation (e.g. PIF3); while others

regulate only PHYA-mediated inhibition of hypocotyl elon-

gation (e.g. HFR1; Fairchild et al., 2000; Fankhauser and

Chory, 2000; Kim et al., 2003; Koornneef et al., 1980; Soh

et al., 2000). The exact molecular pathway that leads to the

inhibition of hypocotyl elongation is not clear, but microar-

ray analysis suggests that this process involves expressional

changes of various genes related to the cell-wall metabolism

(Ma et al., 2001). However, while the hypocotyl elongation

process is relatively well understood, only a few signalling

components have been identified in other important light

responses, such as seed germination.

Seed germination is regulated by various factors, inclu-

ding abscisic acid (ABA), brassinosteroids (BR), ethylene

and gibberellin (GA; Chiwocha et al., 2005; Debeaujon

and Koornneef, 2000; Kepczynski and Kepczynska, 1997;

Koornneef and van der Veen, 1980; Koornneef et al., 2002;

Leubner-Metzger, 2001; Steber and McCourt, 2001). Among

these, ABA and GA play antagonistic roles. During seed

maturation, ABA levels increase and seed dormancy is

established (Karssen et al., 1983). When the dormant seed is

transferred to conditions favourable for germination, the

level of ABA decreases and de novo GA biosynthesis

commences, disrupting dormancy and triggering germina-

tion (Ogawa et al., 2003). ABA biosynthetic mutants such as

aba1 and aba2 display reduced seed dormancy, while GA

biosynthetic mutants such as ga1 are unable to germinate

even under favourable conditions (Karssen et al., 1983;

Koornneef and van der Veen, 1980; Marin et al., 1996). The

importance of GA during seed germination was further

shown in various GA-signalling mutants. A loss-of-function

mutation in RGL2, a negative regulator of GA responses,

allowed plants to germinate even in the absence of de novo

GA biosynthesis, as did a mutation in SPINDLY, a Ser/Thr

O-linked N-acetyl glucosamine (O-GlcNAc) transferase that

also functions as a negative regulator of GA signalling

(Jacobsen and Olszewski, 1993; Jacobsen et al., 1996; Lee

et al., 2002; Tyler et al., 2004). Light-independent germina-

tion of a ga1 rgl2 rga gai quadruple mutant further suggests

that GAI and RGA, homologues of RGL2, also play roles in

seed germination (Cao et al., 2005). Collectively, these

previous results indicate that ABA and GA biosynthesis

are critical for seed dormancy and seed germination

respectively.

Various external factors, such as light, also play critical

roles in regulating seed germination. The promotion of seed

germination by light was noted as early as the 19th century,

and reversible regulation of lettuce seed germination by red

and far-red light is reported by Borthwick et al. (1952). Later,

studies in Arabidopsis mutants revealed that this reversible

regulation of seed germination by red and far-red light was

determined by phytochromes (Hennig et al., 2002; Shinom-

ura et al., 1994). A direct connection between phytochrome

signalling and de novo GA biosynthesis during seed germi-

nation was demonstrated by inhibition of light-induced seed

germination in the presence of a GA biosynthesis inhibitor;

by the insensitivity of the ga1 mutant to light induction; by

the epistasis of the ga1 mutation to the phyB mutation (Peng

and Harberd, 1997); and by the direct determination of

increased GA levels after light induction (Hilhorst and

Karssen, 1988; Koornneef and van der Veen, 1980; Ogawa

et al., 2003). The increased de novo GA biosynthesis during

light-induced germination of Arabidopsis seeds is due to

increased expression of GA biosynthetic enzymes such as

GA 3b-hydroxylase (Yamaguchi et al., 1998). It seems likely

that some of the phytochrome-interacting proteins trans-

duce light signals, leading to activation of GA biosynthetic

gene transcription during seed germination.

We showed previously that PIF3-Like 5 (PIL5/PIF1/

bHLH015), a phytochrome-interacting bHLH protein, is a

key negative regulator of seed germination, and that light

promotes seed germination partly by inhibiting the function

of PIL5 (Oh et al., 2004). Here we demonstrate that PIL5

mediates seed germination by simultaneously regulating

the expression of GA biosynthetic and catabolic genes, and

also show that light inhibits PIL5 function by activating the

phytochrome-mediated degradation of the PIL5 protein.

Results

PIL5 regulates seed germination through gibberellin

Our previous work showed that PIL5 is a key negative

regulator in PHY-mediated promotion of seed germination

(Oh et al., 2004). As phytochromes are known to promote

seed germination by activating de novo GA biosynthesis, we

examined whether PIL5 negatively regulates seed germina-

tion by repressing de novo GA biosynthesis (Yamaguchi

et al., 1998). We first tested the effects of paclobutrazol, an

inhibitor of GA biosynthesis, on the ability of the pil5 mutant

to germinate irrespective of light conditions.

For the PHYB-dependent germination assay, we irradiated

seeds with a far-red light pulse and transferred them directly

to the dark with or without a second irradiation with a red

light pulse. The far-red light pulse inactivated PHYB and

PIL5 regulates seed germination via GA 125

ª 2006 The AuthorsJournal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 47, 124–139

other type II phytochromes by converting them to the Pr

form, while the subsequent red light pulse-activated PHYB

and other type II phytochromes by converting them to the

Pfr form. As PHYB is the major photoreceptor responsible

for promoting seed germination under these experimental

conditions (Shinomura et al., 1994), the seeds fail to germi-

nate when PHYB is either mutated or inactivated by a far-red

pulse, but germinate when PHYB is activated by a red light

pulse. As shown in Figure 1(a), wild-type Arabidopsis

Columbia (Col-0) germinated only when PHYB was activated

by the red light pulse, while the pil5 mutant germinated

irrespective of red or far-red light irradiation. However,

treatment with paclobutrazol blocked germination of both

wild-type and pil5 mutant seeds, even under continuous

white light irradiation. These results indicate that de novo

GA biosynthesis is necessary for germination of both wild-

type and pil5 mutant seeds under PHYB-dependent germi-

nation assay conditions.

Arabidopsis seed germination is also promoted by PHYA if

the phytochrome is allowed to accumulate during a pro-

longed imbibition (Ohet al., 2004; Shinomuraet al., 1996). To

test whether de novo GA biosynthesis is also required for the

role of PIL5 in the PHYA-dependent germination, we irradi-

ated seeds with a far-red pulse to inactivate the type II

phytochromes, then imbibed the seeds for 48 h to allow

accumulation of PHYA. After imbibition, seeds were trans-

ferred to darkness either directly or after a second irradiation

with far-red light. To exclude the effect of PHYB, we used a

phyB mutant and a pil5 phyB double mutant for the experi-

ment. As shown in Figure 1(b), the phyB mutant did not

germinate if transferred directly to darkness, but germinated

if the imbibed seeds were irradiated with far-red light. In

contrast, the pil5 phyB double mutants germinated partially

even if transferred directly to darkness, and the rate increased

further by the irradiation of far-red light. None of the tested

seeds germinated in the presence of paclobutrazol. These

results collectively suggest that de novo GA biosynthesis is

also required for PHYA-dependent germination.

To further prove that PIL5 regulates seed germination

through GA, we generated a pil5 ga1 double mutant and

examined the rate of seed germination. GA1 encodes ent-

copalyl diphosphate synthase, which converts geranylgera-

nyl diphosphate to ent-copalyl diphosphate during GA

biosynthesis. The ga1 mutant is devoid of de novo GA

biosynthesis, and seed germination does not occur in this

mutant under all light conditions unless exogenous GA is

supplied (Koornneef and van der Veen, 1980). Our germina-

tion assay showed that both the ga1 mutant and the pil5 ga1

double mutant failed to germinate under all light conditions,

while the pil5 mutant germinated irrespective of light

conditions (Figure 2). These results indicate that the ga1

mutation is epistatic to the pil5 mutation for seed germina-

tion in the absence of exogenous GA, suggesting that GA

biosynthesis is a target for the action of PIL5.

Figure 1. Paclobutrazol inhibits germination of pil5 mutant seeds.

(a) Germination percentages of Col-0 and pil5-1 mutant seeds under PHYB-

dependent germination conditions either in the presence (PAC) or absence (N)

of paclobutrazol. Upper diagrams depict light treatment schemes for the

experiments. Rp, a red-light pulse (20 lmol m)2 sec)1) for 5 min after a far-

red light pulse (3.2 lmol m)2 sec)1) for 5 min. FRp, a far-red light pulse

(3.2 lmol m)2 sec)1) for 5 min. WLc, continuous white light (80–

100 lmol m)2 sec)1).

(b) Germination percentages of the phyB-9 and pil5-1/phyB-9 mutants under

PHYB-dependent germination conditions in the presence (PAC) or absence

(N) of paclobutrazol. Upper diagrams depict light treatment schemes for the

experiments. No, imbibition without far-red light irradiation. FRi, imbibition

followed by far-red light irradiation (3.2 lmol m)2 sec)1) for 4 h. Error bars

indicate standard deviations.

126 Eunkyoo Oh et al.


Our observation that the ga1 mutation is epistatic to the pil5

mutation, coupled with our finding that paclobutrazol

inhibits germination of the pil5 mutant, suggested that

PIL5 inhibits seed germination by inhibiting GA biosynthe-

sis. To test this hypothesis, we examined whether applica-

tion of exogenous GA could overcome the inhibitory effect

of PIL5 overexpression on seed germination by treating

wild-type and various mutant seeds with GA and determin-

ing the germination percentages.

We first determined the amount of GA required to

promote seed germination in the ga1 mutant. We used

GA4, a bioactive form of GA, for a gibberellin, and deter-

mined the germination percentages in the PHYB-dependent

germination assay when seeds were treated with GA

concentrations ranging from 0 to 10 lM. As shown in

Figure 3(a), over 10 lM GA was sufficient to induce near

100% seed germination in the ga1 mutant. To ensure a

sufficient GA supply, 10 lM GA was used for the following

experiments unless otherwise indicated.

As shown in Figure 3(b), wild-type seeds germinated

following a pulse with red light, but failed to germinate with

far-red light alone. When exogenous GA was supplied, all

wild-type seeds germinated even if only a far-red light pulse

was applied. Consistent with the notion that PHYB is the

major photoreceptor under these germination conditions,

the untreated phyB mutant failed to germinate under all light

(a)

(b)

(c)

Figure 3. Germination percentages retain light dependency in the presence

of exogenous GA.

(a) Determination of GA concentration needed for the rescue of germination

defects in the ga1 mutant under PHYB-dependent germination assay condi-

tions.

(b) Germination percentages of Col-0, pil5-1, phyB-9 and pil5-1/phyB-9 seeds

under PHYB-dependent germination conditions in the presence of 10 lM GA

(GA4) or the absence of GA (N). Upper diagrams, abbreviations as in Figure 1.

Error bars, standard deviations.

(c) Light-dependent germination percentages of ga1 and pil5/ga1 mutant

seeds in the presence of exogenous GA. Light treatment schemes and

notations are as described in (b).

Figure 2. The ga1 mutation is epistatic to the pil5 mutation in seed germi-

nation.

Germination percentages were examined in the ga1, pil5-1 and pil5-1/ga1

mutants under PHYB-dependent germination conditions. Upper diagrams,

abbreviations as in Figure 1.



conditions. In the presence of exogenous GA, the phyB

mutant seeds germinated irrespective of light conditions,

but the germination percentage was around 40% that of the

GA-treated wild-type seeds. The lower germination percent-

age of the phyB mutant was not due to an intrinsic defect in

germination, as 100% of phyB mutant seeds germinated

under continuous light (data not shown). GA treatment also

substituted for red light in the pil5 phyB double mutant. The

pil5 mutant germinated irrespective of light conditions, and

the pil5 phyB double mutant germinated partially in the

absence of exogenous GA. When supplied with exogenous

GA, the pil5 phyB double mutant seeds germinated near

100%, irrespective of light conditions (Figure 3b). The lower

germination percentage of far-red treated pil5 phyB double

mutant than that of far-red treated pil5 mutant is probably

due to the presence of other components and the functional

role of PHYB, either in dry seeds or in maturing seeds, as

discussed previously (Oh et al., 2004). Consistent with this,

the lower expression levels of GA biosynthetic genes were

observed in the pil5 phyB double mutant compared with the

pil5 single mutant (Figure S2c).

The lower germination percentage of the phyB mutant

compared with wild type in the presence of the same

amount of exogenous GA suggests that light may regulate

seed germination through additional processes other than

de novo GA biosynthesis. To investigate this possibility, we

examined whether the germination percentage of the ga1

mutant showed light dependency in the presence of exo-

genous GA. If de novo GA biosynthesis was the sole light-

regulated process, the germination percentage of GA-trea-

ted ga1 mutants should not show any light dependency,

because GA cannot be synthesized endogenously in the ga1

mutant. However, as shown in Figure 3(c), the germination

percentage of the ga1 mutant was light-dependent. Mutant

seeds supplemented with 1 lM GA4 showed nearly 95%

germination when irradiated with red light, but only about

40% germination when irradiated with far-red light. Treat-

ment with higher concentrations of GA reduced this light

dependency, but the trend seemed to indicate that light

regulates seed germination not only through de novo GA

biosynthesis, but also through other processes. Unlike the

ga1 single mutant, the pil5 ga1 double mutant showed no

light dependency of germination, suggesting that PIL5 plays

an important role in the additional processes other than GA

biosynthesis (Figure 3c).

We then examined whether exogenous GA could over-

come the inhibitory effect of PIL5 overexpression on seed

germination. As shown in Figure 4(a), the PIL5 overexpres-

sion lines [PIL5OX1 and PIL5OX3 (Myc-tagged PIL5)] failed

to germinate even after red light irradiation, but GA treat-

ment increased their germination percentages in a concen-

tration-dependent manner. In addition, the rates of GA-

induced germination in the PIL5OX lines were inversely

related to the expression levels of PIL5, and red light was

more effective than far-red light in promoting seed germi-

nation in the presence of exogenous GA (Figure 4a). In

PIL5OX3, the germination percentage was nearly 100%

when red light was irradiated in the presence of 10 lM

GA4, but 70% when far-red light was irradiated with the

same GA treatment. Collectively, these results indicate that

PIL5 inhibits seed germination not only through de novo GA

biosynthesis, but also through other processes.

(a)

(b)

Figure 4. Exogenous GA overcomes the inhibitory effect of PIL5 overexpres-

sion on PHYB-dependent seed germination.

(a) Germination percentages of PIL5OX1 and PIL5OX3 seeds were examined

under PHYB-dependent germination conditions by varying exogenously

supplied GA. Upper diagrams, abbreviations as in Figure 1. Error bars,

standard deviations. RT-PCR data show expression levels of PIL5 in the

PIL5Oxs seeds. Expression of ubiquitin (UBQ) was used as a loading control.

(b) Additive regulation of seed germination by light and exogenous GA in

PIL5OX1 and PIL5OX3 seeds in the presence of 10 lM GA (GA4) or the absence

of GA (N). After the irradiation of a far-red pulse, seeds were irradiated by red

light (20 lmol m)2 sec)1) for various durations (0–12 h).



As we previously reported that the PIL5OX lines required a

longer irradiation time for germination, we next examined

whether GA treatment altered the required irradiation time

for germination (Oh et al., 2004). We tested the germination

percentages of PIL5OX seeds in the presence or absence of

GA coupled with red light irradiations of various durations.

As shown in Figure 4(b), 5 min red light irradiation was

sufficient to fully induce germination in wild-type seeds,

while much longer irradiation times were required for the

PIL5OXs. Wild-type seeds germinated even in the absence of

red light irradiation if exogenous GA was supplied. In the

case of PIL5OX3, GA treatment reduced the required irradi-

ation time to achieve 100% germination from 720 to 5 min,

indicating that the negative role of PIL5 on seed germination

is overcome additively by light and GA.

As PIL5 is also a negative regulator of PHYA-dependent

seed germination, we investigated whether GA treatment

could overcome the inhibitory role of PIL5 on PHYA-

dependent seed germination by determining the germina-

tion percentages of wild-type and various mutant seeds in

the presence or absence of exogenous GA. In the absence of

GA, the germination percentages of wild-type seeds in-

creased in response to longer far-red light irradiation times,

while the phyA mutant failed to germinate regardless of

irradiation time (Figure 5). In the case of the PIL5OX lines,

increased far-red light irradiation times increased the ger-

mination percentages especially in the weaker line (PI-

L5OX3). In the presence of GA, both wild-type and phyA

mutant seeds germinated well, even in the absence of far-

red light irradiation (Figure 5). In the case of PIL5OX seeds,

the germination percentage increased further if GA was

supplied. Taken together with the inhibition of PHYA-

dependent seed germination by paclobutrazol, these results

indicate that PIL5 regulates seed germination negatively

through GA also under PHYA-dependent seed germination

conditions.

PIL5 regulates both GA synthesis and GA degradation

PHY-mediated light signalling induces de novo GA biosyn-

thesis by increasing the expression of two GA 3b-hydroxy-

lases that convert biologically inactive GA9 to biologically

active GA4 (Yamaguchi et al., 1998). As PIL5 is a phyto-

chrome-interacting protein that negatively regulates seed

germination through GA, we investigated the expression of

GA-biosynthesis genes in both the pil5 mutant and PIL5OX

seeds (Oh et al., 2004). For the experiment, various mutant

seeds were irradiated with a far-red pulse with or without a

subsequent red light pulse and incubated in the dark for 12 h

before sampling. This experimental condition is identical to

the PHYB-dependent germination assay condition, except

that seeds were sampled for expression analysis 12 h after

irradiation.

We first examined the expression of two representative

marker genes (EXP1 and CP1, encoding expansin 1 and

cystein proteinase 1 respectively) that are induced by GA

during seed germination (Ogawa et al., 2003; Yamauchi

et al., 2004). The wild-type seeds showed induced marker

gene expression when PHYB was activated by a red light

pulse (inductive condition), but not when PHYB was inacti-

vated by a far-red light pulse (non-inductive condition)

(Figure 6). The pil5 mutant seeds, which germinated irres-

pective of light conditions, showed increased expression of

two marker genes irrespective of light conditions. The

PIL5OX seeds, which failed to germinate under all light

conditions, showed lower levels of EXP1 and CP1 irrespect-

ive of light conditions. These results indicated that the

expression patterns of the two GA-inducible marker genes

are consistent with the germination patterns of the various

mutants.

We next determined the expression of two GA 3b-

hydroxylase genes (GA3ox1 and GA3ox2). As shown in

Figure 6, both GA3ox1 and GA3ox2 were expressed in

wild-type seeds following irradiation with red light but not

far-red light. In the pil5 mutant, GA3ox1 and GA3ox2

expression was constitutively high, irrespective of light

conditions. In contrast, no GA3ox1 and GA3ox2 expres-

sion was detected in the PIL5OX mutants, irrespective of

light conditions. Overall, the expression patterns of

GA3ox1 and GA3ox2 were very similar to those of the

two GA-inducible marker genes, collectively suggesting

that PIL5 negatively regulates seed germination, at least

partly by repressing the expression of GA biosynthetic

genes.

Figure 5. Exogenous GA overcomes the inhibitory effect of PIL5 overexpres-

sion on PHYA-dependent seed germination.

Germination percentages of Col-0, pil5-1, phyA-211, pil5-1/phyA-211, PIL5OX1

and PIL5OX3 seeds under PHYA-dependent germination conditions in the

presence of 10 lM GA (GA4) or in the absence of GA (N). After irradiation of a

far-red light pulse, seeds were imbibed for 48 h, then irradiated by far-red

light (3.2 lmol m)2 sec)1) for 0, 4 or 12 h. Error bars, standard deviations.



Our germination analyses indicated that both light and

PIL5 regulate seed germination through additional proces-

ses other than GA biosynthesis. One candidate process is

the degradation of biologically active GA. During seed

germination, the level of a bioactive GA shows an initial

rapid increase and reaches a subsequent plateau concom-

itant with the increase of an inactive GA, suggesting that

both synthesis and degradation of biologically active GA

occur during seed germination (Ogawa et al., 2003). Thus it

is possible that PIL5 inhibits seed germination not only by

repressing the expression of GA biosynthetic genes, but also

by activating the expression of GA catabolic genes.

To investigate this possibility, we examined the GA2ox

genes, which encode the GA 2-oxidases responsible for

converting biologically active GA4 and/or its precursors to

biologically inactive forms. Of the eight GA2ox genes

present in the Arabidopsis genome, we focused on the

expression of GA2ox2, because it was expressed at high

levels and its expression was light-dependent in germina-

ting seeds (data not shown). As shown in Figure 6(a),

GA2ox2 was highly expressed in seeds that failed to

germinate: far-red light-irradiated wild-type seeds, and both

red and far-red light-irradiated PIL5OX seeds. In contrast,

GA2ox2 was expressed at low levels in seeds that germina-

ted well: red light-irradiated wild-type seeds, and both red

and far-red light-irradiated pil5 mutant seeds. Taken

together, these results indicate that PIL5 negatively regu-

lates the biosynthesis of bioactive GA in germinating seeds,

not only by repressing GA3ox1 and GA3ox2, but also by

activating the expression of GA2ox2.

Although GA2ox2 was expressed at high levels in PIL5OX

mutant seeds, irrespective of light, the expression level of

GA2ox2 was higher in far-red light-irradiated PIL5OX seeds

than in red light-irradiated PIL5OX seeds (Figure 6a). This

light-dependent expression of GA2ox2 in the PIL5OX seeds

might partly explain the light-dependent germination per-

centage of PIL5OX in the presence of exogenous GA

(Figure 4a). Similarly, light-dependent germination was also

observed in the ga1 mutant in the presence of exogenous

GA (Figure 3c), although the pil5 ga1 double mutant did not

show any light dependency. To investigate whether light-

dependent expression of GA2ox2 is also partly responsible

for the light-dependent germination percentages of these

mutants, we determined the expression of GA2ox2 in the

ga1 mutant and the pil5 ga1 double mutant. As shown in

Figure 6(b), GA2ox2 expression was higher in the far-red

light-irradiated ga1 mutant seeds than in the red light-

irradiated ga1 mutant seeds. In the pil5 ga1 double mutant,

GA2ox2 expression was slightly higher in the far-red light-

irradiated seeds, but the overall expression level was lower

than that of far-red light-irradiated ga1 seeds. As GA 2-

oxidase degrades biologically active GAs, these results

suggest that the light-dependent expression of GA2ox2

partly explains the light-dependent germination percent-

ages of PIL5OXs and ga1 mutant seeds in the presence of

exogenous GA (Thomas et al., 1999).

To investigate the relationship between PIL5 and PHYB in

seed germination, we determined the expression levels of

GA3ox1, GA3ox2 and GA2ox2 in the phyB mutant and in

the pil5 phyB double mutant. Under PHYB-dependent

(a)

(b)

(c)

Figure 6. PIL5 regulates GA biosynthesis by repressing the expression of GA

3b-hydroxylase genes (GA3ox1 and GA3ox2) and by activating the expression

of a GA 2-oxidase gene (GA2ox2) under PHYB-dependent germination

conditions (for quantitive RT-PCR data see Figure S2).

(a) Expression patterns of GA3ox1, GA3ox2, GA2ox2, EXP1 and CP1 in Col-0,

pil5-1 and PIL5OX1 seeds under PHYB-dependent germination conditions.

Upper diagrams, abbreviations as in Figure 1. Expression of S18 was used as

a loading control. Germination percentages indicated below figure.

(b) Light-dependent expression of GA2ox2 in ga1 and pil5/ga1 mutant seeds.

Light treatment scheme and notations are as described in (a).

(c) Expression patterns of GA3ox1, GA3ox2 and GA2ox2 in Col-0, pil5-1,

PIL5OX1, phyB-9 and pil5-1/phyB-9 seeds.



germination assay conditions, the phyB and PIL5OX seeds

failed to germinate, while the pil5 phyB double mutant

germinated only partially (Figure 3b). Consistent with these

findings, seeds that germinated under the assay conditions

(wild type and pil5 mutant) showed increased GA3ox1 and

GA3ox2 expression levels, but decreased expression levels

of GA2ox2 (Figure 6c), while seeds that failed to germinate

(PIL5OX and phyB mutant) showed decreased GA3ox1 and

GA3ox2 expression and increased GA2ox2 expression. The

pil5 phyB double mutant showed intermediate expression

levels of both GA biosynthetic and GA catabolic genes,

which is consistent with its intermediate germination per-

centage. These data indicate that both PHYB and PIL5

regulate bioactive GA levels during seed germination via

mediating both synthesis and degradation of GA. As PIL5 is

a PHYB-interacting, downstream-signalling component,

these data further suggest that PHYB promotes seed germi-

nation partly by inhibiting the negative role of PIL5 in the

biosynthesis of bioactive GA.

As PIL5 is also a negative regulator of seed germination

under PHYA-dependent germination conditions, we investi-

gated whether PIL5 regulates the expression levels of

GA3ox1, GA3ox2 and GA2ox2 under these conditions. For

the experiment, far-red-irradiated seeds were imbibed for

48 h to allow the accumulation of PHYA. After imbibition,

far-red light was applied again to activate PHYA, and the

seeds were incubated for 8 h in the dark before sampling.

Under the PHYA-dependent germination assay conditions,

wild-type, pil5 mutant and pil5 phyA double mutant seeds

germinated following far-red irradiation, but PIL5OX and

phyA mutant seeds did not. Consistent with our findings

under PHYB-dependent germination conditions, the GA-

inducible marker genes (EXP1 and CP1) were highly

expressed in seeds that germinated under PHYA-dependent

germination conditions, but were expressed at low levels in

seeds that did not germinate (Figure 7a). The seeds that

germinated showed higher expression levels of GA3ox1 and

GA3ox2, but lower expression levels of GA2ox2. In contrast,

the seeds that failed to germinate showed lower expression

levels of GA3ox1 and GA3ox2, but higher expression levels

of GA2ox2 (Figure 7a,b). These data indicate that PIL5

regulates both GA synthesis and degradation under PHYA-

dependent germination conditions. Our data suggest that

PHYA promotes seed germination partly by inhibiting the

negative role of PIL5 on the biosynthesis of bioactive GA,

being similar to the relationship observed between PIL5 and

PHYB. In addition, the higher expression of GA biosynthetic

genes in the pil5 single mutant compared with the pil5 phyA

double mutant after inductive FR irradiation further suggests

the presence of other factors that mediate PHYA signalling

to GA biosynthesis.

Our expression analysis of GA biosynthetic genes

suggested that the amount of bioactive GA was likely to

be increased in the pil5 mutant and decreased in PIL5OX

plants. To confirm this, we irradiated various mutant

seeds with far-red pulses with or without subsequent red

light pulses, and quantified the GA4 contents. As shown

in Figure 8, the GA4 level was twofold higher in red light-

treated wild-type seeds versus far-red light-treated wild-

type seeds. In the pil5 mutant, GA4 levels were similar to

those found in red-light treated wild-type seeds, regard-

less of light conditions. In PIL5OX plants, the GA4 level

(a)

(b)

Figure 7. PIL5 regulates GA biosynthesis by repressing the expression of GA

3b-hydroxylase genes (GA3ox1 and GA3ox2) and activating the expression of

a GA 2-oxidase gene (GA2ox2) under PHYA-dependent germination condi-

tions (for quantitive RT–PCR data see Figure S3).

(a) Expression patterns of GA3ox1, GA3ox2, GA2ox2, EXP1 and CP1 in Col-0,

pil5-1 and PIL5OX1 seeds under PHYA-dependent germination conditions.

After irradiation of a far-red light pulse (3.2 lmol m)2 sec)1), seeds were

imbibed for 48 h, then irradiated with far-red light (3.2 lmol m)2 sec)1) for 4 h

(FR) and transferred to the dark, or left unirradiated (N) and transferred to the

dark. Expression of S18 was used as a loading control. Germination

percentages indicated below figure.

(b) Expression patterns of GA3ox1, GA3ox2 and GA2ox2 in Col-0, pil5-1,

PIL5OX1, phyA-211 and pil5-1/phyA-211 seeds.



was slightly lower than that found in far-red-treated wild-

type seeds, regardless of light conditions. The higher

contents of bioactive GA in pil5 mutants and the lower

contents of bioactive GA in the PIL5OX plants are

consistent with the expression patterns of GA biosynthetic

and catabolic genes (Figure 6). Taken together, our data

indicate that PIL5 negatively regulates seed germination

by lowering bioactive GA levels.

Our expression analysis of GA2ox2 gene suggested that

GA catabolic activity should be lower in the pil5 mutant

and higher in the PIL5OX. As GA9 and GA4 are catabolized

to GA51 and GA34, respectively, by GA2ox, we determined

levels of two GA catabolic products (GA34 and GA51)

(Table S1). Consistent with the lower expression of

GA2ox2 gene in red light-treated wild-type seeds, levels

of two GA catabolic products were higher in far-red light-

treated seeds than those in red light-treated wild-type

seeds. In the pil5 mutant, the GA catabolic products were

similar to those of red light-treated wild-type seeds

regardless of light conditions. In contrast, levels of GA

catabolic products were very low in the PIL5OX. As the

level of catabolic products depends on substrate availab-

ility, we determined the ratio of GA catabolic products

and their substrates (GA9 and GA4) and found that the

ratio is lower in the pil5 mutant and higher in PIL5OX

(Table S1). Taken together, these results indicated that GA

catabolic activity is lower in the pil5 mutant and higher in

PIL5OX.

PIL5 protein is degraded by both red and far-red light

irradiation

Previous reports indicated that PIF3, another phytochrome-

interacting bHLH protein, is degraded through the 26S pro-

teasome by red and far-red lights (Bauer et al., 2004; Park

et al., 2004). The degradation of PIF3 by light partly explains

how phytochromes inhibit the function of PIF3 in certain

light responses. PIL5 is very similar to PIF3 in terms of its

amino acid sequence, its ability to interact with phyto-

chromes, and its functional relationship with phytochromes

(Huq et al., 2004; Oh et al., 2004). Thus it is tempting to

speculate that phytochromes inhibit PIL5 by activating the

degradation of PIL5 during seed germination. To determine

whether PIL5 protein is degraded by red and far-red light, we

generated transgenic Arabidopsis plants expressing a Myc-

tag fused PIL5 under the control of the CaMV 35S promoter.

The Myc-tagged PIL5 was found to be functional, and one

Myc-tagged PIL5 overexpression line (PIL5OX3) was chosen

for use in the following Western blot experiments.

As the primary role of PIL5 is regulating seed germination,

we examined whether red or far-red light regulates the

stability of PIL5 protein in germinating seeds. Under PHYB-

dependent germination conditions, PIL5 protein was degra-

ded following irradiation of seeds with a pulse of red light,

but not far-red light (Figure 9a). However, after imbibition

both red and far-red light effectively triggered degradation

of PIL5 protein (Figure 9b). Similar degradation of PIF1/PIL5

protein by light has recently been reported in seedlings

(Shen et al., 2005). These results suggest that the degrada-

tion of PIL5 protein, like PIF3 protein, is also activated by

phytochromes in germinating seeds.

To provide additional evidence that phytochromes are

photoreceptors for the activation of PIL5 degradation, we

determined the degradation of PIL5 protein in the phyA and

phyB mutants. As shown in Figure 9(c), PIL5 was degraded

by far-red light under PHYA-dependent germination condi-

tions in the wild-type background but not in the phyA mutant

background (Figure 9c), indicating that PHYA is a main

photoreceptor for the activation of PIL5 degradation in

response to far-red light. In the phyB mutant, the degrada-

tion pattern of PIL5 was slightly more complicated. As

shown in Figure 9(d), the PIL5 protein was initially degraded

more or less equally in both PIL5OX3 and PIL5OX phyB

mutants following irradiation. At 24 h after irradiation, PIL5

protein was still degraded well in PIL5OX, but virtually no

degradation occurred in the PIL5OX phyB mutant. These

results suggested that the degradation of PIL5 is activated by

both PHYB and other type II phytochromes in response to

red light, and the functional PHYB is required for the

prolonged degradation of PIL5 protein after a red-light pulse.

As PIF3 is degraded through the 26S proteasome, we

examined the degradation of PIL5 in the presence of a 26S

proteasome inhibitor (MG132) in seedlings. As shown in

Figure 8. Endogenous GA levels

The pil5 mutant contains increased levels of bioactive GA (GA4), whereas

PIL5OX plants have lower levels of GA4, regardless of light conditions. Upper

diagrams, abbreviations as in Figure 1. Error bar, standard deviation.

Experiments were repeated with similar results using independently incuba-

ted seed sets.



Figures 9(e) and 8(f), the degradation of PIL5 by red and far-

red lights was greatly reduced in the presence of MG132,

suggesting that PIL5 is also degraded through the 26S

proteasome both under red and far-red lights. MG132 had a

much weaker effect on PIL5 degradation in seeds, when

compared with seedlings, possibly due to lower permeation

by this chemical (Figure S1). Taken together, our results

indicate that phytochromes act as photoreceptors to activate

the degradation of PIL5 protein through the 26S proteasome.

Discussion

We report here that PIL5, a phytochrome-interacting bHLH

protein, negatively regulates seed germination by repress-

ing two GA 3b-hydroxylases genes (GA3ox1 and GA3ox2)

and activating a GA 2-oxidase gene (GA2ox2) at the tran-

scriptional level. Repression of GA biosynthetic genes and

activation of GA catabolic genes by PIL5 leads to increased

levels of bioactive GA in the pil5 mutant and decreased

levels of bioactive GA in PIL5OX seeds, regardless of light

conditions. Our functional relationship studies further

suggest that phytochromes promote seed germination by

inhibiting PIL5, at least partly via activation of PIL5 protein

degradation through the 26S proteasome. Based on these

results, we propose a photogermination model in which

PIL5 plays a negative role in the biosynthesis of bioactive

GA, and PIL5 degradation by phytochromes is activated in

response to inductive light signals (Figure 10).

GA synthesis and degradation are regulated during

phytochrome-mediated promotion of seed germination

De novo GA biosynthesis plays a critical role in the phyto-

chrome-mediated promotion of seed germination, as shown

by the ability of a GA biosynthetic inhibitor (paclobutrazol)

to inhibit seed germination, and the fact that treatment with

exogenous GA could initiate germination in the absence of

light stimulation (Hilhorst and Karssen, 1988; Yang et al.,

1995). The observation that GA biosynthetic mutants such as

ga1 failed to germinate even after inductive light treatment

further supports the role of GA in the phytochrome-medi-

ated promotion of seed germination (Koornneef and van der

(a) (b)

(d)

(c)

(e) (f)

Figure 9. Phytochromes activate the degrada-

tion of PIL5 protein.

(a) Degradation of PIL5 protein by red or far-red

light under PHYB-dependent germination condi-

tions. PIL5 indicates Myc-tagged PIL5 protein

detected by an anti-Myc antibody. Tubulin (TUB)

was detected by anti-tubulin antibody as a load-

ing control.

(b) Degradation of PIL5 protein by red or far-red

light under PHYA-dependent germination condi-

tions.

(c) Degradation of PIL5 protein in PIL5OX3 and

PIL5OX3/phyA mutant seeds by far-red light

under PHYA-dependent germination conditions.

(d) Degradation of PIL5 protein in PIL5OX3 and

PIL5OX3/phyB mutant seeds by red light under

PHYB-dependent germination conditions.

(e) Inhibition of PIL5 degradation by MG132 in

far-red light-irradiated seedlings.

(f) Inhibition of PIL5 degradation by MG132 in red

light-irradiated seedlings. Five DAG dark-grown

seedlings were treated by either DMSO or

MG132 (200 lM), irradiated by red light pulse

for 5 min, and incubated in the dark for 30 min

until harvesting.



Veen, 1980). Previous studies have shown that GA biosyn-

thesis increases during seed germination due to the tran-

scriptional activation of GA 3b-hydroxylase genes, which

encode enzymes required for the rate-limiting step of GA

biosynthesis (Yamaguchi et al., 1998).

The present work reveals that light regulates the biosyn-

thesis of bioactive GA not only by activating the transcrip-

tion of GA biosynthetic genes, but also by suppressing the

transcription of at least one GA catabolic gene. Expression of

two GA 3b-hydroxylase genes (GA3ox1 and GA3ox2)

increased dramatically on the perception of inductive light

signals, whereas expression of a GA 2-oxidase gene

(GA2ox2) decreased under inductive conditions and in-

creased under non-inductive conditions. The net effect of

this reciprocal regulation of biosynthetic and catabolic

genes is probably a sharper increase or decrease of bioactive

GA levels, compared with regulation of either alone. This

reciprocal regulation was found to be under the control of

phytochromes. Under PHYB-dependent germination assay

conditions, induction of the GA 3b-hydroxylase genes and

repression of the GA 2-oxidase gene were abolished in the

phyB mutant. Similarly, under PHYA-dependent germina-

tion assay conditions, this reciprocal regulation was abol-

ished in the phyA mutant. These data indicate that both

PHYA and PHYB promote the biosynthesis of bioactive GA

during seed germination by activating expression of GA 3b-

hydroxylase genes and repressing the expression of at least

one GA 2-oxidase gene.

We further found that PIL5 plays a critical role for this

reciprocal regulation of GA synthesis and catabolism

during phytochrome-mediated promotion of seed germi-

nation. We previously showed that PIL5 is a phytochrome-

interacting bHLH protein that preferentially interacts with

the Pfr forms of both PHYA and PHYB. Our characteriza-

tions of mutant and overexpression lines revealed that

PIL5 is a key negative regulator for both PHYA- and PHYB-

dependent germination processes (Oh et al., 2004). In the

present work we found that expression of GA3ox1 and

GA3ox2 is increased in the pil5 mutant and decreased in

PIL5OX seeds, indicating that PIL5 represses expression of

these GA biosynthetic genes. Similar higher expression of

GA3ox1 and GA3ox2 in the pil5 mutant have been

reported recently by Penfield et al. (2005). In contrast,

GA2ox2 is expressed at lower levels in the pil5 mutant

and at higher levels in PIL5OX seeds, indicating that PIL5

activates expression of this GA catabolic gene. The

expression patterns of GA3ox1, GA3ox2 and GA2ox2 in

the phyB, pil5 phyB, phyA and pil5 phyA mutants further

indicated that PIL5 reciprocally regulates these genes

during phytochrome-mediated promotion of seed germi-

nation. The consequences of GA biosynthetic and cata-

bolic gene-expression patterns are reflected in the levels

of bioactive GA in germinating seeds. Consistent with the

higher expression of GA biosynthetic genes and the lower

expression of GA catabolic genes, the levels of bioactive

GA were increased and decreased in the pil5 and PIL5OX

plants, respectively, regardless of light conditions. Taken

together, these data indicate that PIL5 is a key negative

component that links phytochrome signals to the expres-

sion of GA biosynthetic and catabolic genes during seed

germination.

Although our finding that light signals trigger induction

of GA 3b-hydroxylase genes is consistent with the previ-

ous report, the relationship between phytochromes and

these genes observed in the present study differs slightly

from the earlier findings (Yamaguchi et al., 1998). The

previous study showed that PHYB controlled GA3ox2

expression, while GA3ox1 expression was controlled by

other phytochromes. However, our data clearly indicate

that these genes were not expressed in the phyB mutant

under the PHYB-dependent germination assay conditions,

showing that the expression of both GA3ox1 and GA3ox2

are both controlled by PHYB (Figure S2c). We further

showed that PHYA also regulates the expression of both

GA3ox1 and GA3ox2 under PHYA-dependent germination

assay conditions. The basis of these discrepancies is not

immediately clear, but may be related to the use of

different Arabidopsis ecotypes. The previous reports were

Figure 10. Proposed model for the phytochrome-mediated promotion of

seed germination in Arabidopsis.

PIL5, a phytochrome-interacting bHLH protein, inhibits seed germination by

repressing the transcription of GA 3b-hydroxylase genes and activating the

transcription of a GA 2-oxidase gene. Under inductive light conditions,

phytochromes activate the degradation of PIL5 through the 26S proteasome,

thus increasing the biosynthesis of bioactive GAs. Bioactive GAs promote

seed germination by activating the degradation of GAI, RGA and RGL2

through SCFSLY1,SNE. For simplicity, other factors implicated in mediating

light signalling to GA biosynthesis and the degradation of GA9 to GA51 are not

indicated in the model.



based on analyses of the Landsberg erecta (Ler) ecotype,

while we utilized the Col-0 ecotype in the present work.

Differences have been reported in light signalling and

seed germination between the Ler and Col ecotypes,

suggesting the possibility that light-signalling processes

such as expression of GA3ox1 might differ between these

ecotypes (Borevitz et al., 2002; van der Schaar et al., 1997;

Wolyn et al., 2004; Yanovsky et al., 1997). It will be

interesting to determine if any of the quantitative trait

loci between Ler and Col are related to the expression of

GA biosynthetic genes, such as GA3ox1. Alternatively, the

use of slightly different protocols in the PHYB-dependent

germination assays might account for the differences

between our results and those of other groups.

It is not yet known how PIL5 reciprocally regulates the

transcription of GA biosynthetic and catabolic genes. Many

transcription factors have been shown to activate one set of

genes while repressing another, although the molecular

mechanisms for these reciprocal regulations vary (Holm

et al., 2002; Monte et al., 2004). For example, PIL5 may

activate transcription of two different downstream transcrip-

tion factors, one that activates GA2ox2 transcription and one

that represses expression of GA3ox1 and GA3ox2. Alternat-

ively, PIL5 may regulate these genes directly, with its activity

as an activator or a repressor depending on the context

created by promoters or binding cofactors. Further analysis

will be required to elucidate the reciprocal regulation of

these genes by PIL5, leading to greater understanding of

light signalling in general.

Phytochromes inhibit PIL5 by promoting its protein

degradation

Protein degradation plays a key role in PHY-mediated light

responses. Mutations in the COP/DET/FUS genes cause

constitutive photomorphogenic phenotypes, even in the

dark, and analysis of these genes indicated that the

majority of them encode proteins involved in protein

degradation (Wei and Deng, 1996). Among these, COP1

encodes a RING-finger type ubiquitin E3 ligase; COP10

encodes a ubiquitin E2 conjugation enzyme; and most of

the others encode subunits of a regulatory complex sim-

ilar to the 19S lid complex of the 26S proteasome (von

Arnim and Deng, 1993; Suzuki et al., 2002; Wei and Deng,

1996). In the light-signalling process, HFR1, HY5 and LAF1,

three positive light-signalling components, are ubiquiti-

nated by COP1 in the dark, and the ubiquitinated compo-

nents are subsequently degraded by 26S proteasome

(Duek et al., 2004; Jang et al., 2005; Osterlund et al., 2000;

Seo et al., 2003; Yang et al., 2005). Photoreceptors such as

phytochromes or cryptochromes inhibit the E3 ligase

activity of COP1 partly by excluding COP1 from the nuc-

leus (von Arnim and Deng, 1994). Thus photoreceptors

potentiate light responses that are mediated by HFR1, HY5

and LAF1 by inhibiting the degradation of these factors by

COP1.

Phytochromes also modulate the stability of directly

interacting components. PIF3, a bHLH protein, was the first

identified phytochrome-interacting protein (Ni et al., 1998).

The physiological function of PIF3 is complex, depending on

the light responses examined. For hypocotyl elongation, it

acts as a negative component in PHYB- but not PHYA-

mediated inhibition of hypocotyl elongation. For anthocy-

anin synthesis, PIF3 acts as a positive component in PHY-

mediated accumulation of anthocyanin; in terms of chloro-

plast development, PIF3 plays a positive role during the

transition from dark to light (Kim et al., 2003; Monte et al.,

2004). Expression analysis indicated that the level of PIF3

transcript is not significantly affected by light, whereas PIF3

protein is rapidly degraded by red or far-red light (Baueret al.,

2004; Park et al., 2004). This degradation is activated by PHYA

in response to far-red light, and by PHYB and PHYD in

response to red light. In both cases, PIF3 is degraded by

ubiquitination followed by degradation via the 26S protea-

some.

We show here that phytochromes also activate the

degradation of PIL5, another phytochrome-interacting

bHLH protein, through the 26S proteasome. Similarly to

PIF3, PIL5 regulates various light responses, including

seed germination, hypocotyl elongation, shoot gravitro-

pism and protochlorophyllide biosynthesis (Huq et al.,

2004; Oh et al., 2004). Genetically, phytochromes inhibit

the function of PIL5 in those PHY-mediated light

responses. Thus our data, showing the degradation of

PIL5 by red or far-red light, demonstrate a mechanism by

which phytochromes inhibit the function of PIL5 to initiate

those light responses.

Our work, and the previous analyses, show that phyt-

ochromes are able to regulate the degradation of various

light-signalling components through at least two different

pathways. One pathway acts through COP1, which leads

to degradation of largely positive components such as

HFR1, HY5 and LAF1 in the dark (Duek et al., 2004; Jang

et al., 2005; Osterlund et al., 2000; Seo et al., 2003; Yang

et al., 2005). The regulation of COP1 activity by phyto-

chromes is partly due to the exclusion of COP1 from the

nucleus under light conditions (von Arnim and Deng,

1994). However, HFR1 accumulates more quickly under

far-red light irradiation than COP1 exclusion proceeds,

indicating that this exclusion may not be the sole

regulatory mechanism (Yang et al., 2005). The other

pathway, acting through a yet-unknown mechanism,

degrades directly interacting, largely negative components

such as PIF3 and PIL5 through the 26S proteasome under

light conditions (Bauer et al., 2004; Park et al., 2004).

Further investigations will be required to determine how

phytochromes activate the degradation of their directly

interacting proteins in the light.



Experimental procedures

Plant materials and growth conditions

Arabidopsis thaliana plants were grown in a growth room with a 16-h light/8-h dark cycle at 22–24�C for general growth and seed har-vesting. The ga1 mutant was obtained from the Arabidopsis StockCenter (Salk_109115; Alonso et al., 2003). For generation of the PI-L5OX3 transgenic line, the full-length PIL5 cDNA was amplified withspecific primers (5¢-AGA GTG ATC AAA AAT GCA TCA TTT TGT CCCTGA C-3¢ and 5¢-AGA GTG ATC ACC ACC TGT TGT GTG GTT TCC-3¢),cloned into a pBI-HTM vector for expression of a fusion proteinbearing both His and Myc tags, and transformed into wild-typeplants (Col-0) by Agrobacterium-mediated transformation. Threeindependent homozygous lines (PIL5OX3, PIL5OX4 and PIL5OX5)were established. As they all showed phenotypes consistent withthe previously established PIL5OX1 and PIL5OX2 lines expressingthe native PIL5 gene, we used PIL5OX3 seeds for our experimentswhere indicated. All plants used in the experiments (pil5-1, phyA-211, phyB-9, ga1, PIL5OX1 and PIL5OX3) were Col-0 ecotype back-ground. Different mutants used in each figure were grown at thesame time, in the same growth room, in the same tray. Seeds werestored at 22�C in white paper bags.

Germination assay

For the PHYB-dependent germination assay, triplicate sets of 60seeds for each mutant were surface sterilized and plated on aqueousagar medium (0.6% phytoagar, pH 5.7). At 1 h after the start of seedsterilization, the plated seeds were irradiated with red(20 lmol m)2 sec)1) or far-red (3.2 lmol m)2 sec)1) light for 5 min.After 5 days’ incubation in the dark, germinated seeds were deter-mined by the emergence of radicles. For the PHYA-dependent ger-mination assay, the plated seeds were first irradiated with far-redlight (3.2 lmol m)2 sec)1) for 5 min, incubated in the dark for 48 h,then further irradiated with far-red light (3.2 lmol m)2 sec)1) forvarious times. Four days later, the germinated seeds were counted.To determine the effect of paclobutrazol on seed germination, trip-licate sets of 60 seeds for each mutant were spotted on agar platescontaining 100 lM paclobutrazol, incubated under PHYA- and PHYB-dependent germination assay conditions, and counted for germi-nated seeds. For the germination assay in the presence of exogen-ous GA, triplicate sets of 60 seeds for each mutant were spotted onagar plates containing various concentrations of GA4 (0–10 lM).

Gene-expression analysis

A total of 50 ll seeds were plated on wet filter paper and incubatedunder PHYA- or PHYB-dependent germination assay conditions,with the exception that the plates were incubated for 12 h beforeseeds were harvested for expression analysis. Total RNA wasextracted from the seeds using an Ambion RNA extraction kitaccording to the manufacturer’s guidelines (Ambion, Austin, TX,USA).

The following primers were used for RT–PCR analysis of geneexpression: GA3ox1 (5¢-CCG AAG GTT TCA CCA TCA CT-3¢ and 5¢-CCC CAA AGG AAT GCT ACA GA-3¢); GA3ox2 (5¢-TAG ATC GCA TCCCAT TCA CA-3¢ and 5¢- TGG ATA ACT GCT TGG GTT CC-3¢); GA2ox2(5¢-TGA CTC GGT TAG AGC AGG AG-3¢ and 5¢-CTT GAA CCT CCCGTT AGT CA-3¢); EXP1 (5¢-TCA CAT GTC AAT GGT TAC GC-3¢ and 5¢-TGT CCC CAG TTT CTT GAC AT-3¢); CP1 (5¢-TGA TGA GTC CAT CATCAA CG-3¢ and 5¢-TGT AGG ATT TGT CGC AGT CA-3¢); S18 (5¢-CCAGCG ATC GTT TAT TGC TT-3¢ and 5¢-AGT CTT TCC TCT GCG ACC

AG-3¢); PIL5 (5¢-GGG GAT TTT AAT AAC GGT-3¢ and 5¢-GAG ATTATG AAC TTC AGC AGC ACG-3¢); UBQ (5¢-GAT CTT TGC CGG AAAACA ATT GGA GGA TGG T-3¢ and 5¢-CGA CTT GTC ATT AGA AAGAAA GAG ATA ACA GG-3¢).

Analysis of endogenous GAs

Various mutant seeds were surface sterilized and plated onaqueous agar medium (0.6% phytoagar, pH 5.7). At 1 h after thestart of seed sterilization, the plated seeds were irradiated withred (20 lmol m)2 sec)1) or far-red (3.2 lmol m)2 sec)1) light for5 min. After 26 h incubation in the dark, seeds were collected forGA quantification. Quantitative analysis of GAs was carried out byGC-selected ion monitoring (SIM) using 2H-labelled GAs as inter-nal standards, as described previously (Gawronska et al., 1995).Briefly, a pre-purified ethyl acetate-soluble fraction containingGAs was subjected to HPLC purification using a reverse-phasecolumn (Capcell Pak C18 SG120; Shiseido Fine Chemicals, Tokyo,Japan). When necessary, GA-containing fractions after reverse-phase HPLC were further purified through another round of HPLCusing an ion-exchange column (Senshu Pak N[CH3]2, 1151-N;Senshu Scientific, Tokyo, Japan) to ensure the removal ofimpurities for reliable quantification. The purified fractions weresubjected to GC–SIM analysis using a mass spectrometer (Auto-mass Sun; JEOL, Tokyo, Japan) equipped with a GC (6890N;Agilent Technologies, Palo Alto, CA, USA) and a capillary column(DB-1; Agilent Technologies) after derivatization. Authentic GAsamples and 2H-labelled internal standards were purchasedfrom Professor Lewis Mander (Australian National University,Canberra).

Protein extraction and protein gel blotting

A total of 50 ll seeds were plated on wet filter paper, incubatedunder PHYA- or PHYB-dependent germination assay conditions,and harvested at the times indicated. The harvested seeds wereground in liquid nitrogen and solubilized in buffer (100 mM NaH2-

PO4, 10 mM Tris–Cl, 8 M urea pH 8.0) by vigorous vortexing. Thedebris was cleared by centrifugation at 20 000 g for 10 min at 4�C.Western blot analyses were performed as described by Park et al.(2004) to determine the PIL5 protein levels. The Myc tag was used toassess PIL5 protein levels with an anti-Myc antibody. To determinethe effects of MG132, five DAG dark-grown seedlings were pre-treated by either DMSO or MG132 (200 lM) and incubated undervarious light conditions until harvesting.

Acknowledgements

We thank Ms Masayo Sekimoto and Mr Atushi Hanada (RIKEN PlantScience Center) for GA analysis. This work was supported in part bygrants from the KRF (C00044), KOSEF (R21-2003-000-10002-0), thePlant Diversity Research Center of the 21st Frontier Research Pro-gram (PF0330508-00), and the Plant Metabolism Research Centerfunded by KOSEF.

Supplementary Material

The following supplementary material is available for this articleonline:Figure S1. The inhibition of PIL5 degradation by MG132 requires aprolonged pretreatment in seeds. The degradation of PIL5 without(A) or with (B) 24 hours pretreatment.



Figure S2. Quantitive RT-PCR data corresponding to Figure 6.Figure S3. Quantitive RT-PCR data corresponding to Figure 7.Table S1 (A) Endogenous GA levels (ng/g dry seed) in germinatingseeds of Col-0, pil5, and PIL5OX1. Two sets of data were obtainedfrom independently incubated seed samples. (B) Ratio of GAcatabolic products and their substrates ([GA34 þ GA51]/[GA4 þ GA9])This material is available as part of the online article from http://www.blackwell-synergy.com

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