Activation of the heterotrimeric G protein α-subunit GPA1 ...sippe.ac.cn/gh/2009 Annual Report/84.pdf · ted into the cell by heterotrimeric G proteins composed of a, b and c subunits

Activation of the heterotrimeric G protein a-subunit GPA1suppresses the ftsh-mediated inhibition of chloroplastdevelopment in Arabidopsis

Lingang Zhang1,†, Qing Wei1,†, Wenjuan Wu1,†, Yuxiang Cheng1, Guangzhen Hu1, Fenhong Hu1, Yi Sun1, Ying Zhu1,

Wataru Sakamoto2 and Jirong Huang1,*

1National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for

Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China, and2Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan

Received 29 November 2008; revised 29 January 2009; accepted 10 February 2009; published online 16 March 2009.*For correspondence (fax +86 21 5492 4015; e-mail [email protected]).†These authors contributed equally to this work.

SUMMARY

Heterotrimeric G protein knock-out mutants have no phenotypic defect in chloroplast development, and the

connection between the G protein signaling pathway and chloroplast development has only been inferred

from pharmaceutical evidence. Thus, whether G protein signaling plays a role in chloroplast development

remains an open question. Here, we present genetic evidence, using the leaf-variegated mutant thylakoid

formation 1 (thf1), indicating that inactivation or activation of the endogenous G protein a-subunit (GPA1)

affects chloroplast development, as does the ectopic expression of the constitutively active Ga-subunit

(cGPA1). Molecular biological and genetic analyses showed that FtsH complexes, which are composed of type-

A (FtsH1/FtsH5) and type-B (FtsH2/FtsH8) subunits, are required for cGPA1-promoted chloroplast develop-

ment in thf1. Furthermore, the ectopic expression of cGPA1 rescues the leaf variegation of ftsh2. Consistent

with this finding, microarray analysis shows that ectopic expression of cGPA1 partially corrects mis-regulated

gene expression in thf1. This overlooked function of G proteins provides new insight into our understanding of

the integrative signaling network, which dynamically regulates chloroplast development and function in

response to both intracellular and extracellular signals.

Keywords: chloroplast development, heterotrimeric G proteins, variegation, FtsH protease, Arabidopsis.

INTRODUCTION

Chloroplasts are essential for plant cells because they not

only harbor photosynthesis but also make a wide range of

fundamental molecules (Neuhaus and Emes, 2000). Chlo-

roplast development, accompanied by biosynthesis of

chlorophylls, and the subsequent assembly of photosyn-

thetic apparatus in the thylakoid membrane, is completed

within a few hours of light perception (Kleffmann et al.,

2007). It is known that chloroplast development is dyna-

mically regulated by intracellular developmental cues,

retrograde signaling from chloroplasts to nuclei and the

ever-changing environmental conditions (Mullet, 1988; Nott

et al., 2006). Although remarkable progress has been made

towards understanding the molecular machinery that

controls chloroplast biogenesis (Aldridge et al., 2005), the

integrative network that senses intra- and extracellular

signals, and subsequently transfers these signals to the

machinery, remains poorly understood.

Leaf variegation mutants are useful for dissecting this

signaling network, because the severity of variegation is

frequently dependent upon environmental conditions and

developmental stages (Sakamoto, 2003; Aluru et al., 2006).

To date, a number of nuclear genes, including FtsH5/VAR1,

FtsH2/VAR2, VAR3 and THF1, have been shown to control

leaf variegation in Arabidopsis (Yu et al., 2007). Analysis of

these mutants demonstrates that variegation is caused by

abnormalities in various chloroplast functions (Sakamoto

et al., 2003; Aluru et al., 2006). FtsH protease, which belongs

to the ATP-dependent AAA+ protein family, and forms a

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The Plant Journal (2009) 58, 1041–1053 doi: 10.1111/j.1365-313X.2009.03843.x

84

hexameric ring structure, is the best-characterized example

for explaining the mechanism of variegation in Arabidopsis

(Sakamoto, 2006). FtsH subunits are encoded by type-A

(FtsH1 and FtsH5) and type-B (FtsH2 and FtsH8) genes. The

genes of the same type are functionally redundant, and

plants defective in either type do not survive (Sakamoto

et al., 2003; Yu et al., 2005).

Function of FtsH complexes has been shown to repair the

photodamaged photosystem-II (PSII) reaction center D1

protein and to control chloroplast development (Lindahl

et al., 2000; Bailey et al., 2002; Sakamoto et al., 2003; Zalts-

man et al., 2005; Yoshioka et al., 2006). It is proposed that a

threshold level of the FtsH complex is required for normal

chloroplast function: chloroplast function is impaired, and

then white or yellow sectors form when the complex levels

fall below the threshold (Yu et al., 2007).

In eukaryotes, many extracellular signals are sensed by

heptahelical cell-surface receptors, designated as G protein-

coupled receptors (GPCR). These signals are then transmit-

ted into the cell by heterotrimeric G proteins composed of a,

b and c subunits (Neves et al., 2002). In the canonical model

of G-protein signaling, G proteins are activated by binding

ligands to their corresponding receptors, which subse-

quently catalyze the replacement of guanosine diphosphate

(GDP) with guanosine triphosphate (GTP) on the Gbc-

complexed Ga subunit. Then, both the GTP-bound Ga and

released Gbc can transfer signals to downstream effectors.

The signaling is switched off by the intrinsic GTP hydrolysis

activity of Ga, because the GDP-bound Ga has low affinity

for effectors, but high affinity for Gbc. The duration of G-

protein signal transduction can be significantly shortened by

regulators of G-protein signaling (RGS), through accelerat-

ing the GTP hydrolysis of Ga (Siderovski et al., 1996;

Siderovski and Willard, 2005). This G-protein signaling

system, composed of GPCRs, heterotrimeric G proteins

and effectors, and RGS proteins, is present in the plant

kingdom (Temple and Jones, 2007). In Arabidopsis, only

single canonical a (GPA1) and b (AGB1) subunits, and two c(AGG1 and AGG2) subunits, have been identified. The other

important components, GPCR and RGS, are also few (Chen

et al., 2003; Gookin et al., 2008). Several downstream effec-

tors or interactors, including a cupin domain-containing

protein (PRN1), phospholipase Da1 (PLDa1), prephenate

dehydratase 1 (PD1) and thylakoid membrane formation 1

(THF1) have been shown to bind physically to GPA1 (Lapik

and Kaufman, 2003; Zhao and Wang, 2004; Huang et al.,

2006; Warpeha et al., 2006), whereas only one genetically

interacting protein, a Golgi-localized hexose transporter

SGB1 (suppressor of G protein b1), has been shown for

AGB1 (Wang et al., 2006).

The involvement of G proteins in chloroplast develop-

ment was first demonstrated by pharmaceutical studies with

hypocotyl cells of the tomato aurea mutant, which is almost

totally deficient in active phytochrome A (PhyA) (Neuhaus

et al., 1993; Bowler et al., 1994). It was concluded that the G

protein is sufficient to transduce PhyA signaling through

second messages, cyclic GMP (cGMP) and Ca2+/calmodulin.

Signaling pathways mediated by cGMP and Ca2+/calmodulin

coordinately promote the expression of photosynthesis-

related proteins, and the assembly of photosynthetic com-

plexes. Several reports also indicated that G proteins

modulate plastid gene expression, which may affect

chloroplast development and function (Romero and Lam,

1993; Dhingra et al., 2004; Warpeha et al., 2007). However,

whether G-protein signaling plays a role in chloroplast

development remains an open question. Here, using the

leaf-variegated mutants thf1 and ftsh2 we provide several

lines of evidence supporting the hypothesis that G proteins

finely modulate chloroplast development, at least in part, via

the regulation of FtsH gene expression in Arabidopsis.

RESULTS

GPA1 is a positive regulator for chloroplast development

in thf1

Previous studies showed that THF1 physically interacted

with GPA1 (Huang et al., 2006), and that the null thf1 mutant

had a variegated phenotype in both cotyledons and leaves

(Wang et al., 2004). Here, we found that the gpa1thf1 double

mutant displayed a more severe leaf-variegated phenotype

than the thf1 single mutant. We therefore studied whether

the G-protein signaling pathway was involved in chloroplast

development. As RGS proteins accelerate the deactivation of

the Ga subunit, the RGS1 mutation might reduce the degree

of variegation in the thf1 background. Indeed, the rgs1 thf1

plants produced fewer yellow sectors than the thf1 plants,

whereas the thf1 gpa1 double mutant consistently displayed

more severe variegation than thf1 at both seedling and

mature plant stages (Figures 1a,b, S1). Leaf chlorophyll

content was in agreement with the severity of the variega-

tion phenotype among the mutants (Figure 1c). The rgs1

thf1 leaves had 18.9% more chlorophyll than the thf1 leaves,

whereas the thf1 gpa1 leaves had only 75.8% of the thf1 level

of chlorophyll. These data indicate that GPA1 is a positive

regulator for chloroplast development in thf1.

Ectopic expression of cGPA1 rescues the thf1 variegation

phenotype

To further study a role of G-protein signaling in chloroplast

development, thf1 plants were transformed with a consti-

tutively activated (GTPase-deficient) form of GPA1Q222L,

designated as cGPA1. The results showed that ectopic

expression of cGPA1 in thf1 (cGPA1/thf1) clearly rescued the

variegation phenotype of thf1 in both cotyledons and leaves

(Figure 2a). The chlorophyll content of the cGPA1/thf1 lines

was comparable with that of wild-type (WT) plants, whereas

1042 Lingang Zhang et al.

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85

thf1 accumulated 83.3% of the WT chlorophyll level in leaves

(Figure 2b). In addition, the ratio of chlorophyll a to b (Chl a:b)

in thf1 (2.82) was also reduced to the WT level (2.50) by cGPA1

(Figure 2c). Thus, we concluded that the ectopic expression

of cGPA1 was able to rescue the thf1 variegation phenotype.

Ectopically expressed cGPA1 in thf1 affects chloroplast

ultrastruture and PSII activity

The observations described above prompted us to investi-

gate the effect of cGPA1 on chloroplast development and

photosynthesis in thf1. We examined the chloroplast ultra-

structure in green sectors of mature leaves from 43-day-old

WT, thf1 and cGPA1/thf1 plants, and in the yellow sectors

of thf1 leaves, using transmission electron microscopy. In

green sectors, most of the chloroplasts in thf1 and cGPA1/

thf1 transgenic leaves had similar ultrastructures, including

grana and stroma thylokoids, as those observed in the WT

(Figure 3a,b,c). However, in cGPA1/thf1 mesophyll cells,

about 20% of the chloroplasts had unusual granal stacks

(Figure 3d). Such a chloroplast was not observed in both WT

and thf1 leaves under our growth conditions. It is well known

that PSII and the associated main chlorophyll a/b light-

harvesting complex (LHCII) are predominantly localized in

the stacked thylakoid membrane, which is essential for

optimizing the photosynthetic machinery under various

environmental conditions (Anderson, 1999). The observed

change in chloroplast ultrastructure from cGPA1/thf1 leaves

may indicate a role of the G-protein signaling pathway in

photosynthesis under environmental stress. In yellow sec-

tors of thf1 leaves, plastids in most mesophyll cells lacked

organized thylakoid structures, and contained many mem-

brane vesicles (Figure 3e). Interestingly, chloroplasts were

present in some spongy cells that contacted with the lower

epidermis in the thf1 yellow sectors (Figure 3f,g), but had

fewer thylakoid membranes (Figure 3h). Taken together, our

ultrastructural data support the hypothesis that G-protein

signaling is involved in the regulation of chloroplast

development.

A previous study demonstrated that thf1 mutants were

hypersensitive to excessive light stimuli (Keren et al., 2005).

To determine whether the ectopic expression of cGPA1

could rescue the sensitivity of the thf1 plant to high

intensities of light, we measured the PSII maximum photo-

chemical efficiency (Fv/Fm; Fv, variable fluorescence; Fm,

maximum fluorescence) in green regions. No difference in

Fv/Fm was detected among WT, thf1 and cGPA1/thf1 plants

grown in normal light conditions (70–100 lmol m)2 s)1;

Figure 3i, left bars). However, cGPA1/thf1 transgenic

(a)

(c)

(b)

Figure 1. GPA1 is a positive regulator for chloroplast development in thf1.

(a) Comparison of 10-day-old wild-type (WT), gpa1, rgs1, thf1, gpa1 thf1 and

rgs1 thf1 seedlings.

(b) Comparison of 43-day-old thf1, gpa1 thf1 and rgs1 thf1 plants.

(c) Leaf chlorophyll content of the plants in (b). Data were means of three

independent experiments �SD (with six plants in each experiment). *Signif-

icant difference (P < 0.05) between the WT and mutants; FW, fresh weight.

(a)

(b) (c)

Figure 2. The ectopic expression of cGPA1 rescues the thf1 variegation

phenotype.

(a) Representative photographs of wild-type (WT), thf1 and two independent

transgenic plants. Upper panel, 10-day-old seedlings; lower panel, 43-day-old

plants.

(b) and (c) Chlorophyll content and ratio of chlorophyll a to b (Chl a:b) in 43-

day-old WT, thf1, cGPA1/thf1-A and cGPA1/thf1-B plants, respectively. The red

arrow indicates a white or yellow sector of thf1 leaves. Data were means of six

independent experiments with �SD (each experiment contained six plants).

*Significant difference (P < 0.05) between the WT and thf1 mutant; Chl,

chlorophyll; FW, fresh weight.

G proteins and chloroplast development 1043


86

plants were more resistant to high light intensity

(1500 lmol m)2 s)1) than were thf1 plants, but were more

sensitive than the WT plants (Figure 3i, middle and right

bars). This result indicates that the ectopic expression of

cGPA1 can partially recover PSII activity in thf1 leaves.

THF1 mutation leads to a decrease in FtsH protease that is

partially recovered by the ectopic expression of cGPA1

Based on the above results, we proposed that the positive

action of cGPA1 on chloroplast development and PSII

activity in the thf1 background might be associated with

some proteins in thylakoid membranes, where the photo-

synthetic complexes are localized. To test this hypothesis,

we isolated thylakoid membranes from WT, thf1 and cGPA1/

thf1 leaves. The membrane proteins were fractionated by

SDS-PAGE (Figure 4a left panel). The result indicated that

the ectopic expression of cGPA1 in thf1 obviously enhanced

the density of a band with a molecular weight of approxi-

mately 71 kDa (marked with an asterisk in Figure 4a).

Quantitative analysis indicated that the relative level of the

band in cGPA1/thf1 was about 60% higher than that in thf1

(Figure 4a right panel). To identify this band, the gel frag-

ment was excised, digested with trypsin and analyzed by

matrix-associated laser desorption ionization time-of-flight

(MALDI-TOF) mass spectrometry (Figure 4b). Sequencing of

the polypeptides showed that the band contained FtsH iso-

forms, including FtsH1 and FtsH5 (Figure 4c). This finding

provided a line of important evidence to suggest that the

variegation phenotype of thf1 may be attributed to a low

level of the FtsH complex.

As the D1 protein, a core subunit of the PSII reaction

center, is a substrate for FtsH protease, we measured the

activity of FtsH protease in thf1 by examining the stability of

the D1 protein. To do this, thylakoid membranes were

extracted from leaves that were treated with Lincomycin (an

inhibitor of protein biosynthesis in chloroplasts) for 1 h, and

then subsequently exposed to high levels of light. As shown

in Figure 5a, the D1 protein was more stable in thf1 than in

the WT during treatment, whereas the stability of D2 protein,

which is another subunit of the PSII reaction center, and is

used as a control, was not affected by the THF1 mutation.

This result suggests that FtsH activity is reduced in thf1.

We then investigated FtsH levels by Western blot analysis.

Note that FtsH levels detected by antibodies against FtsH5

and FtsH2 represent type A and type B, respectively

(Sakamoto et al., 2003). The result indicated that both type-

A and tyep-B FstHs were significantly reduced in thf1

(Figure 5b). Consistent with this finding, the ectopic expres-

sion of cGPA1 in thf1 enhanced the accumulation of FtsH

subunits (Figure 5b). Quantitative analysis showed that the

relative levels of type-A and type-B FtsHs in cGPA1/thf1

accumulated to levels that were 2- and 1.35-fold that found in

thf1, respectively. However, expression of THF1 was not

altered in any of the ftsh single mutants (Figure 5c). Thus,

these results suggest that THF1 functions as a regulator for

FtsH expression.

The ectopic expression of cGPA1 enhances mRNA levels

of FtsH subunits

The reduced accumulation of FtsHs in thf1 could be a result

of either reduced expression or increased degradation, or of

both. To verify these possibilities, the steady-state levels of

mRNAs for FtsHs were analyzed by Northern blot with

(a)

(c)

(e)

(g)

(i)

(h)

(f)

(d)

(b)

Figure 3. Chloroplast ultrastructure and light sensitivity of wild-type (WT),

thf1 and cGPA1/thf1 plants. Representative photographs of chloroplast

ultrastructure in green (a–d) and yellow (e–h) sectors of 43-day-old leaves.

(a) WT, (b) thf1 and (c) cGPA1/thf1 have a similar ultrastructure.

(d) cGPA1/thf1 shows a large granal stack.

(e) Plastids with many membrane vesicles.

(f) Chloroplasts in spongy cells contacting the lower epidermis.

(g) An enlarged image of the boxed portion of (f).

(h) Chloroplast ultrastucture in spongy cells.

(i) Light sensitivity of 43-day-old plants measured as the maximum photo-

system-II (PSII) photochemical efficiency (Fv/Fm) after exposure to light

(1500 lmol m)2 s)1) for 1.5 or 3 h. Data were means � SD (n = 10). Scale

bars: 1 lm (a–e,h); 10 lm (f,g). The black arrows point to granal thylakoids,

and the white arrow points to stroma thylokoids. Asterisk(s) indicate statistical

differences between thf1 and cGPA1/thf1 (*P < 0.05; **P < 0.01).



87

specific probes. Our data showed that expression of all four

FtsH genes in thf1 was similar to that in the WT (Figure 6a),

suggesting that the THF1 mutation-induced decrease in FtsH

accumulation takes place at a post-transcriptional step. In

contrast, the ectopic expression of cGPA1 in thf1 increased

the level of each FtsH transcript. In particular, the expression

of FtsH2 was 2.3-fold higher than that in the WT (Figure 6a).

This result suggests that the recovered level of FtsHs in

cGPA1/thf1 is at least in part attributable to an increase in

FtsH transcripts.

We then investigated the stability of FtsH subunits in thf1.

To do this, FtsH levels in thylakoid membranes were

dynamically examined after protein biosynthesis in the

cytoplasm was inhibited by cycloheximide (CHX). Immuno-

blot analysis showed that 50% or less of FtsHs remained in

thf1 and cGPA1/thf1, whereas about 90% of FtsHs remained

in WT plants when treated with CHX for 6 h (Figure 6b).

These data indicated that the ectopic expression of cGPA1

had no effect on the thf1-induced instability of FtsH

complexes.

Genetically, THF1 and FtsHs act in the same pathway

for leaf variegation

The critical role FtsH protease played in the thf1-induced

variegation phenotype prompted us to investigate whether

they functioned in the same pathway for leaf variegation.

Homozygous mutants of thf1 were crossed with ftsh5 or

ftsh2. New phenotypes with severely variegated or albino

seedlings were observed in the F2 generation. The albino

seedlings, which mimic the phenotypes of ftsh5 ftsh1 and

ftsh2 ftsh8 double mutants (Zaltsman et al., 2005), were

(a)

(b)

(c)

Figure 4. SDS-PAGE profiles of thylakoid mem-

brane proteins and MALDI-TOF MS analysis.

(a) A representative SDS-PAGE profile stained

with Coomassie blue (left panel). The 71-kDa

band is indicated by an asterisk. The relative

levels of the 71-kDa band were normalized to the

band marked with two asterisks (right panel).

(b) MALDI-TOF MS of tryptic peptides eluted

from the 71-kDa band in (a). Asterisks indicate

peaks matching the peptide fingerprints of FtsH

peptides.

(c) A representative product ion spectra of

m/z 1508.75; the sequence derived is shown as

an inset.



88

identified as ftsh5 thf1 and ftsh2 thf1 double mutants (Fig-

ure 7a). The genotyping analysis indicated that plants with

severe variegated phenotypes were homozygous for thf1,

and were heterozygous for either ftsh5 or ftsh2, which was

more apparent than in any of the thf1, ftsh5 and ftsh2 single

mutants (Figure 7a,b). These results suggest that the THF1

mutation has a strong effect on FtsH function. To evaluate

the effect of FtsH1 and FtsH8 on thf1 variegation, we made

ftsh1 thf1 and ftsh8 thf1 double mutants. As shown in Fig-

ure 7c, both the ftsh1 thf1 and ftsh8 thf1 double mutants

survived, and displayed phenotypes that were similar to that

of the thf1 mutant. This result is in accord with previous

reports showing that neither the single ftsh1 and ftsh8

mutants nor the double ftsh1 ftsh8 mutant displayed leaf

variegation (Zaltsman et al., 2005).

To determine directly whether the FtsH complex is a

bottleneck limiting chloroplast development in thf1, we

tested if type-A or type-B subunits were able to complement

the variegation phenotype of thf1. Because FtsH genes

within the same type are interchangeable, the full-length

cDNA of FtsH1 or FtsH8 was introduced into the thf1 mutant

(Figure S2a). As expected, overexpression of either FtsH1 or

FtsH8 markedly rescued the variegated or yellow cotyledons

of thf1 (Figure 7d, upper panel). The leaf variegation of thf1

was also largely complemented by FtsH1 and FtsH8 (Fig-

ure 7d, lower panel). This result demonstrates that FtsH

protease plays an important role in the THF1-regulated

development of chloroplasts.

Chloroplast development promoted by the ectopic

expression of cGPA1 is dependent on FtsH complexes

Given that the cGPA1-promoted development of chloroplast

was attributable to the elevated levels of the FtsH complex,

(a)

(b)

(c)

Figure 5. The THF1 mutation leads to a decrease in FtsH accumulation, which

is partially rescued by the ectopic expression of cGPA1.

(a) FtsH activity was measured by D1 stability. The D2 level was used as a

control. +Linc, leaves were floated on 1 mM lincomycin solution at

20 lmol m)2 s)1 for 3 h. +Linc/HL, leaves that had been pre-treated with

lincomycin were subsequently exposed to 1800 lmol m)2 s)1 irradiance;

2.0 lg of chlorophyll were loaded in each lane.

(b) Western blot analysis of FtsH proteins in 43-day-old wild-type (WT), thf1

and cGPA1/thf1 plants (left upper panel). The left lower panel shows the

relative levels of type-A and type-B in thf1 and cGPA1/thf1, compared with the

WT. The error bars represent�SD. The gel stained with Coomassie blue (right

panel) was shown as a loading control.

(c) Western blot analysis of THF1 in 10-day-old WT, ftsh5, ftsh2, ftsh1, ftsh2

and thf1 plants. *Non-specific band detected by the antibody.

(a)

(b)

(c)

Figure 6. Analyses of FtsH mRNA levels and protein stability.

(a) Northern blot analysis of the expression of four FtsH in 43-day-old wild-

type (WT), thf1 and cGPA1/thf1 plants (left panel). The relative mRNA levels

are shown in the right panel. All values were normalized to rRNA levels, and

the WT control was arbitrarily set to 100. Data are means � SD of three

independent experiments.

(b) Immunoblot analysis of FtsH protein stability in 43-day-old WT, thf1 and

cGPA1/thf1 plants. The values below each band represent the density of FtsH5

and FtsH2, respectively, and were normalized to that at 0 h. Leaves were

treated with cyclohexomide for 1 h, and then sampled at the indicated times.

Thylakoid membranes were isolated from these leaves.

(c) The Coomassie blue-stained gel was used as a loading control.



89

the variegation phenotype might also be rescued by the

introduction of cGPA1 into ftsh2. The transgene cGPA1 was

identified by genomic PCR (Figure S2b). Indeed, the ectopic

expression of cGPA1 significantly rescued the variegation

phenotype in the first pair of true leaves, where the varie-

gation is most obvious in ftsh2 mutants (Figure 8a). Western

blot analysis also showed that the transgenic lines accu-

mulated higher levels of FtsH subunits than the ftsh2

mutants (Figure 8b). Quantitative analysis showed that the

relative levels of type-A and type-B FtsHs in ftsh2 only

accumulated to 3.3 and 43.7% of the WT levels, respectively,

whereas in cGPA1/ftsh2 lines the levels were 10.2 and 57.5%

of the WT levels, respectively. Thus, we reasoned that the

type-A and type-B levels, enhanced by the ectopic expres-

sion of cGPA1, were correlated with the recovery of the

severe variegation phenotype of ftsh2.

To further test the hypothesis that the cGPA1-rescued

variegation phenotype was coupled to FtsH subunits, we

incorporated the cGPA1 gene into the ftsh8 ftsh2 double

mutant, by crossing the transgenic cGPA1/ftsh2 line with

the single ftsh8 mutant. The resulting ftsh2 ftsh8 double

mutants displayed white or slightly yellow seedlings (Fig-

ure 8c), whereas the presence of a single wild-type FtsH8

allele in the ftsh2 mutants gradually turned the cotyledons

from yellow to green (Figure 8c). In the F2 population, the

frequency of seedlings with white leaves generated from

ftsh2 · ftsh8 (2.71/16) had no statistical difference from

those from cGPA1/ftsh2 · ftsh8 (2.78/16), suggesting that

the ectopic expression of cGPA1 is incapable of rescuing the

phenotype of the ftsh2 ftsh8 double and FtsH8+/) ftsh2

mutants. A smaller than expected ratio of lethal seedlings (3/

16) was also reported by Zaltsman et al. (2005), because of

some aborted seeds in the siliques of F1 plants. Interestingly,

we found that some of the seedlings with white leaves that

segregated from cGPA1/ftsh2 · ftsh8 exhibited greener cot-

yledons than did those from ftsh2 · ftsh8. We genotyped

seedlings with differentially greening cotyledons and found

that the presence of cGPA1 was correlated with the greener

cotyledons (Figure 8c). In the FtsH8+/) ftsh2 background the

chlorophyll content in cotyledons increased from 0.24 to

0.61 mg g)1 of fresh weight, with the ectopic expression of

cGPA1. Therefore, we conclude that cGPA1-promoted chlo-

roplast development is coupled to the FtsH complex.

Microarray analysis shows that the expression of a batch of

genes in thf1 is recovered by ectopically expressed cGPA1

To better understand the molecular basis underlying

cGPA1-promoted chloroplast development in thf1, we

performed a microarray analysis using total RNAs

extracted from 3-day-old light-grown seedlings of the

cGPA1/thf1 transgenic line, the thf1 mutant and the WT.

Transcriptomic comparisons were performed to find

genes differentially expressed in thf1 over the WT and in

cGPA1/thf1 over thf1 through the genome annotation

program on the TAIR website (http://www.arabidopsis.org/

portals/genAnnotation). Among the 22 743 genes repre-

sented on the Affymetrix chip, 560 genes, including 151

upregulated genes and 409 downregulated genes, were

mis-regulated by at least twofold in thf1 relative to the

WT (Table 1). Consistent with the role of THF1 in chlo-

roplast development, 25.9% (106) of the 409 down-

regulated genes are predicted to function in plastids. This

proportion is markedly higher than that predicted for

whole genomic plastidic proteins, i.e. approximately 3500

(a)

(d)

(b)

(c)

Figure 7. Genetic interaction between THF1 and FtsHs.

(a) Phenotypes of 6-day-old wild-type (WT), thf1, ftsh5, ftsh2, thf1 ftsh5, thf1

ftsh2, FtsH5+/) thf1 and FtsH2+/) thf1 seedlings grown on agar plates. FtsH5+/)

and FtsH2+/) indicate heterozygotes.

(b) Phenotypes of 35-day-old WT, thf1, ftsh5, ftsh2, thf1 FtsH5+/) and thf1

FtsH2+/) plants.

(c) Phenotypes of 10-day-old WT, thf1, ftsh1, ftsh8, thf1 ftsh1 and thf1 ftsh8

seedlings grown on agar plates.

(d) Representative photographs of transgenic plants overexpressing FtsH1

and FtsH8 in thf1, and designated as FtsH1/thf1 and FtsH8/thf1, respectively.

Upper panel, cotyledons of 4-day-old seedlings; lower panel, 35-day-old

transgenic plants.



90

(about 15.4% of the genes in the array; Arabidopsis

Genome Initiative, 2000). In contrast, 10.6% (16) of the 151

upregulated genes encoded proteins targeted to plastids.

On the other hand, we found that 924 genes, including

369 upregulated genes and 555 downregulated genes,

were mis-regulated by at least twofold in the cGPA1/thf1

seedlings compared with the thf1 seedlings (Table 1).

Among these differentially expressed genes in cGPA1/

thf1, compared with thf1, interestingly 18.7% of the

upregulated genes are predicted to function in plastids,

whereas only 6.3% of the downregulated genes are pre-

dicted to function in plastids. These results indicate that

the expression of cGPA1 in thf1 has a significant impact

on the expression profiling of the genes that is directly

attributable to chloroplast development. Furthermore,

among these mis-regulated genes, we found that 49

(32.5%) of the genes upregulated in thf1 were down-

regulated in the cGPA1/thf1 line (Table S1), whereas 32

(7.8%) downregulated genes in thf1 were upregulated in

the cGPA1/thf1 line (Table S2). These opposite expression

(a)

(b)

(c)

Figure 8. The ectopic expression of cGPA1 res-

cues the variegated leaf phenotype of ftsh2.

(a) Phenotypes of 21-day-old wild-type (WT) and

transgenic plants. cGPA1/ftsh2-A and cGPA1/

ftsh2-B are two independent transgenic lines

expressing cGPA1 in ftsh2.

(b) FtsH levels in 10-day-old WT, ftsh2, cGPA1/

ftsh2-A and cGPA1/ftsh2-B plants detected by

immunoblot analysis (left upper panel). The

relative levels of type A and type B in ftsh2 and

two transgenic lines were normalized to those of

WT levels (left lower panel). The right panel

shows the Coomassie blue-stained gel as a

loading control.

(c) Genotype identified by PCR with gene-specific

primers, and comparison of phenotypes of 21-

day-old seedlings with (lanes 1 and 2) or without

(lanes 3 and 4) the transgene of cGPA1 in the

FtsH8+/) ftsh2 (lanes 1 and 3) or ftsh2 ftsh8 (lanes

2 and 4) background.

Table 1 The ectopic expression of cGPA1partially rescues the mis-regulated tran-scripts in thf1: numbers and percentagesof the mis-regulated genes (by at leasttwofold) are shown for thf1 relative to thewild-type (WT), and for cGPA1/thf1 relativeto thf1, respectively

thf1 cGPA1/thf1

Functional categoryUpregulated(151 genes) (%)

Downregulated(409 genes) (%)

Upregulated(369 genes) (%)

Downregulated(555 genes) (%)

Chloroplast/plastid 16 (10.6) 106 (25.9) 69 (18.7) 35 (6.3)Transport 11 (7.3) 31 (7.6) 21 (5.7) 56 (10.1)Defense 13 (8.6) 84 (20.5) 21 (5.7) 116 (20.9)Transcription 14 (9.3) 44 (10.8) 37 (10.0) 37 (6.7)Signal transduction 4 (2.6) 14 (3.4) 9 (2.4) 20 (3.6)Development 7 (4.6) 25 (6.1) 40 (10.8) 23 (4.1)Protein metabolism 14 (9.2) 39 (8.3) 36 (9.7) 39 (7.0)Unknown 43 (28.5) 106 (25.9) 126 (34.1) 145 (26.1)



91

patterns detected in thf1 and cGPA1/thf1 seedlings by the

microarray analysis were validated for selected genes by

Northern bolt analysis (Figure 9a,b). Taken together, the

results indicate that cGPA1 overexpression can partially

correct the mis-regulated gene expression caused by the

THF1 mutation.

DISCUSSION

G-protein signaling pathway for chloroplast development

Chloroplast development must be kept in harmony with

developmental and environmental cues, as photooxidation

easily takes place in developing chloroplasts. The D1 protein

at the reaction center of PSII undergoes rapid renewal, in

which the FtsH protease is required for D1 degradation.

Certainly, a low activity of FtsH protease leads to the accu-

mulation of damaged D1 protein, followed by oxidative

stress and inhibition of chloroplast development or photo-

synthesis. In this study, we established a genetic linkage

between the G-protein signaling pathway and chloroplast

development via FtsH protease. In addition, our data also

indicate that G proteins are probably involved in the

regulation of photosynthesis under various environmental

conditions. In general, our results support a hypothesis

that G proteins are fine tuners, adjusting various cellular

processes to their best status in Arabidopsis.

A role of G proteins in chloroplast development has been

reported in the tomato aurea mutant (Neuhaus et al., 1993;

Bowler et al., 1994). We also explored whether cGPA1 could

rescue the pale-green phenotype of hy2, which is a homolog

of aurea in Arabidopsis (Parks and Quail, 1991; Muramoto

et al., 2005). Unexpectedly, the ectopic expression of cGPA1

was unable to rescue the pale-green phenotype of hy2 (data

not shown), indicating that cGPA1 probably functions spe-

cifically in FtsH-mediated chloroplast development. This

disagreement with pharmaceutical results may reflect dif-

ferent molecular mechanisms underlying G protein-regu-

lated chloroplast development in plant species. The blocked

chloroplast development in hy2 is attributed to the inhibition

of 5-aminolevulinic acid (ALA) biosynthesis (Terry and

Kendrick, 1999).

FtsH protease is regulated by G proteins and THF1

In higher plants, the way in which FtsH protease is regulated

remains unknown, in spite of its critical role in chloroplast

development and photosynthesis. Our several lines of

(a)

(b)

Figure 9. The ectopic expression of cGPA1 par-

tially rescues the mis-regulated transcripts in

thf1. Message RNAs were extracted from 3-day-

old wild-type (WT), thf1 and cGPA1/thf1 seed-

lings that were collected and combined from

three independent experiments. Seedlings were

grown on half-strength MS supplemented with

1% sucrose under short-day conditions.

(a) Northern blot analysis of selected differen-

tially expressed genes in WT, thf1 and cGPA1/

thf1 seedlings. Seedling growth conditions and

the sampling times were the same as described

in Table 1. rRNA is shown as a loading control.

(b) The relative levels of selected gene expres-

sion in (a). The density of each band was

normalized to that of rRNA. The data are means

of three independent experiments. Error bars

represent �SD.



92

evidence showed that FtsH protease is finely controlled by

THF1 and the G-protein signaling pathway. Using genetic

and molecular biological approaches, we found that THF1 is

an important regulator for FtsH protease. Further analysis

indicated that the FtsH complex is not stable in the absence

of THF1. It seems that the THF1-regulated expression of FtsH

is similar to that in the single ftsh2 and ftsh5 mutants, where

the coordinated decrease in FtsH accumulation is attributed

to the degradation of unassembled subunits (Sakamoto

et al., 2003), but not to a reduction in transcriptional levels of

the other FtsH genes (Yu et al., 2004). How FtsH complexes

are post-translationally regulated by THF1 remains to be

further investigated.

Genetic screening for ftsh2 suppressors revealed that the

balance between chloroplast protein synthesis and degra-

dation plays an important role in chloroplast development

(Park and Rodermel, 2004; Miura et al., 2007; Yu et al., 2008).

These suppressors include two subunits (ClpC2 and ClpR1)

of Clp protease, SVR1 (a chloroplast-localized homolog of

pseudouridine synthase), SCO1 (chloroplast elongation fac-

tor G) and FUG1 (chloroplast translation initiation factor 2).

Mutations of these genes ultimately lead to a decrease in

chloroplast protein accumulation. It was proposed that

reduced rates of plastid protein synthesis decrease the

demand for FtsH complexes in developing chloroplasts,

allowing more plastids to overcome a threshold and to turn

green in ftsh2 plants (Yu et al., 2008). In sharp contrast,

cGPA1-promoted chloroplast development is dependent on

an increase in the FtsH expression in the thf1 and ftsh2

backgrounds. In addition, fug1 has been demonstrated to

reduce the photosynthetic activity of ftsh2 as a result of the

suppression of protein biosynthesis in chloroplasts (Miura

et al., 2007), whereas PSII activity in cGPA1/thf1 plants was

higher than in thf1 plants (Figure 3i). Thus, our data reveal a

new mechanism for plants to maintain protein balance in

developing chloroplasts, through the upregulation of gene

expression.

A possible role of G proteins in retrograde signaling

Retrograde signaling from chloroplasts to the nucleus has

recently been shown to mediate the expression of many

nuclear genes in response to a variety of intra- and extra-

cellular changes, such as chlorophyll biosynthesis, high

levels of light and pathogen attack (Marwell et al., 1999; Nott

et al., 2006). However, little is known about how a signal is

transferred from chloroplasts to the nuclei. Retrograde sig-

nals include reactive oxygen species (ROS), intermediates in

chlorophyll biosynthesis, and redox generated by the elec-

tron transfer pathway (Nott et al., 2006; Koussevitzky et al.,

2007). Kato et al. (2007) reported that retrograde signaling

pathways were involved in the suppression of nuclear-

encoded photosynthetic gene expression in the yellow or

white sectors of variegated leaves. Interestingly, G proteins

have been shown to influence cellular responses to oxida-

tive stresses generated in chloroplasts, such as ozone and

far-red irradiation in Arabidopsis (Joo et al., 2005; Wei et al.,

2008). Furthermore, hydrogen peroxide was reported to

promote the dissociation of the Ga subunit from the

approximately 700-kDa complex (Wang et al., 2008). Chlo-

roplasts are recognized as a primary site producing ROS,

and dynamically contact the plasma membrane. As previ-

ously reported, GPA1 and THF1 interact at sites where the

plastid membrane abuts the plasma membrane (Huang

et al., 2006). Thus, whether G proteins are involved in ret-

rograde signaling pathways will be an interesting topic for

future studies.

A model for G protein-mediated chloroplast development

The genetic and molecular biological data presented here

indicate that THF1 functions either upstream of or in parallel

with GPA1 in the regulation of chloroplast development. In

addition, THF1 was also suggested to work downstream of

the GPA1-mediated glucose signaling pathway in roots

(Huang et al., 2006). A plausible explanation for these dif-

ferent results is that G proteins often act in a tissue- or cell-

specific manner, and their multiple functions are executed

by different G-protein complexes (Perfus-Barbeoch et al.,

2004; Chen, 2008). Although the physiological significance

of G protein-mediated chloroplast development remains

unclear, our data lay a solid foundation towards a complete

dissection of the G-protein signaling pathway in the future.

Here, we propose a model for G protein-mediated chlo-

roplast development via FtsH protease (Figure 10). It has

been well documented that FtsH protease is a critical factor

for chloroplast development in Arabidopsis (Yu et al., 2007).

Results derived from this study suggest that THF1 and G

proteins are the new regulators for FtsH protease. THF1 is

highly conserved in oxygenic photosynthetic organisms, but

its biochemical function has not yet been elucidated. THF1 is

Figure 10. A model for G protein-mediated chloroplast development via FtsH

protease. FtsH protease is a key determinant in chloroplast development. In

the present study, we show that mRNAs of both type-A and type-B FtsH genes

are coordinately controlled by G proteins. The THF1-mediated regulation of

chloroplast development may be achieved via G protein-dependent and/or

-independent pathways. THF1 localized on the outer envelope of chloroplasts

may act as the scaffold or modulator that is required for the full activation or

fine control of the G-protein signaling pathway, whereas THF1 in the stroma

or thylakoids regulates the stability of FtsH complexes. Abbreviations:

C, chloroplasts; N, nuclei; PM, plasma membrane.



93

localized in the stroma, thylakoids and the outer envelope

of chloroplasts (Wang et al., 2004; Huang et al., 2006;

L. Zhang and J. Huang, unpublished data). Generally, the

THF1-mediated regulation of chloroplast development may

be achieved via G protein-dependent and/or -independent

pathways. In the G protein-independent pathway, stroma-

and/or thylakoid-localized THF1 controls chloroplast devel-

opment through affecting the stability of FtsH protease.

In the G protein-dependent pathway, the outer envelope-

localized THF1 may function as a scaffold or modulator

required for the full activation or fine control of the G-protein

signaling pathway. The activated form of GPA1 can transmit

signals from the plasma membrane to nuclei via second

messengers and/or other signaling molecules, and ulti-

mately controls the expression of the FtsH genes needed

for chloroplast development. Calcium- and cGMP-depen-

dent pathways have been shown to play an important role in

G-protein signal transduction (Neuhaus et al., 1993; Bowler

et al., 1994). Whether the G protein-regulated FtsH expres-

sion is mediated by Ca2+and/or cGMP signaling pathways

remains to be investigated. The two pathways may crosstalk

to form a complicated mechanism by which the activity of

FtsH protease in chloroplasts is finely regulated at different

levels of gene expression.

EXPERIMENTAL PROCEDURES

Plant materials and growth conditions

The Arabidopsis thaliana ecotype Columbia-0 was used. Themutants used for the analyses or generation of double mutants havepreviously been described: thf1-1 (Huang et al., 2006), gpa1-4(Jones et al., 2003), rgs1-2 (Chen et al., 2003), ftsh1 and ftsh8(Sakamoto et al., 2003), and ftsh2 and ftsh5 (Yu et al., 2004). Doublemutants were identified in the F2 generation by an allele-specificPCR-based genotyping procedure. For phenotypic observation,seeds were stratified at 4�C for 4 days, and then sown onto soil in a22�C growth chamber with an 8-h light/16-h dark cycle. Alterna-tively, surface-sterilized seeds were sown onto half-strength MSagar plates supplemented with 1% sucrose. The seedlings weretransferred to soil once established.

Plasmid constructions and transgenic plants

Plasmids were constructed using the Gateway cloning system(Invitrogen, http://www.invitrogen.com). The constitutively activeform of GPA1(Q222L) was generated as described in Chen et al.(2003). FTSH1 and FTSH8 cDNA were cloned from the ABRC stockU19697 (FtsH1) and U14077 (FtsH8) into the pENTR SD/D-TOPOentry vector with gene-specific primers (Table S3). The fragmentswere then recombined into the pGWB2 destination vector (ResearchInstitute of Molecular Genetics, Shimane University, Japan). Ara-bidopsis plants were transformed by Agrobacterium (GV3101) withthe flower-dip method (Clough and Bent, 1998). Transgenic T1

plants were screened on media supplemented with kanamycin.Transgenic lines were confirmed by RT-PCR or by PCR productsamplified from genomic DNA with gene-specific primers (Table S3).Transgenic lines overexpressing cGPA1 were identified by the

presence of the cGPA1 transgene or transcripts. The PCR productswere further digested with the restriction enzyme DdeI, and gener-ated three bands with 448, 360 and 67 bp.

Chlorophyll content, chlorophyll fluorescence and

transmission electron microscopy

Chlorophyll was extracted with 80% acetone at 4�C for 24 h indarkness. Chlorophyll a and chlorophyll b were determined bythe absorbance at 645 and 663 nm with a spectrophotometer(UNIC UV-2102; PCS, Shanghai, China, http://www.unico1.com.cn/e/index.asp), respectively. Chlorophyll fluorescence was measuredas described by Zhou et al. (2008). Leaves of 43-day-old plantsgrown in short-day conditions (8-h light/16-h dark) were fixed in asolution of 4% glutaraldehyde, and then processed, embedded andviewed via electron microscopy, as described by Harris et al. (1994).

Total RNA extraction and northern blot analysis

Total RNA was isolated using the RNAgents total RNA isolationsystem (Promega, http://www.promega.com) in accordance withthe manufacturer’s instructions. A 10-lg portion of total RNA wasloaded in each lane, separated on formaldehyde gels and thenstained with ethidium bromide to confirm equal loading of RNA.Digoxingenin-labeled DNA probes with gene-specific primers(Table S3) were used in the hybridization solution. Northern blottingwas conducted as described by Huang et al. (2000).

Thylakoid membrane preparation, immunoblot analysis

and protein identification

Thylakoid membrane was prepared according to Aro et al. (1993).Total proteins of thylakoid membranes equivalent to 2.0 lg ofchlorophyll were loaded in each lane, and separated by denaturingSDS-PAGE, and then transferred to Hybond-ECL nitrocellulosemembrane (Amersham Biosciences, now part of GE Healthcare,http://www.gehealthcare.com) and immunoblotted with variousantibodies. The specificity of a-FtsH5 (VAR1) and a-FtsH2 (VAR2) hasalready been examined by Sakamoto et al. (2003). The a-THF1 was agift from Dr Korth (University of Arkansas, http://www.uark.edu).The antibodies AS05084 and AS06146 against the D1 and D2 pro-teins, respectively, were bought from Agrisera (http://www.agrisera.com). Immunodetection of the protein gel blots was performedusing the ECL plus western blotting detection system (AmershamBiosciences). For protein identification, bands were excised fromthe gels and the peptide sequence was analyzed as described byPeltier et al. (2004).

Transcriptomic analysis of genes in cGPA1/thf1 transgenic,

thf1 and WT plants

We used 3-day-old WT (control), thf1 and cGPA1/thf1 seedlings formicroarray analysis. Samples were collected and combined fromthree independent experiments grown on half-strength MS sup-plemented with 1% sucrose under short-day conditions. Total RNAwas isolated using the TRIzol (Invitrogen) extraction procedure, andwas further purified with RNeasy minikits (Qiagen, http://www.qiagen.com). Double-strand cDNA was synthesized using a one-cycle cDNA synthesis kit (Affymetrix, http://www.affymetrix.com),followed by purification on a GeneChip sample clean-up module(Affymetrix). Biotin-labeled cRNA was prepared and hybridized



94

to whole genome Affymetrix ATH1 GeneChip microarrays contain-ing 22 746 Arabidopsis transcripts, as suggested by the manufac-turer. The probe arrays were scanned using the 2500 AffymetrixGeneChip Scanner. The acquired data were analyzed using theMICROARRAY SUITE (MAS 5.0; Affymetrix), and databases of geneexpression profiles were built with single-array analyses (see theAffymetrix statistical algorithms reference guide). The transcripts ofthe whole genome genes were classified into three categories bydetection P-values: present (P < 0.04), absent (P > 0.06) and mar-ginal (0.04 < P < 0.06). The significant difference (P < 0.0025) in theup or downregulated transcriptional level of the detected genesbetween thf1 and the WT or cGPA1/thf1 and thf1 was examinedusing Wilcoxon’s signed rank test. The genes were selected by thesignal log ratio ‡1.0 (with an at least twofold variation of transcriptlevels) for mis-regulated genes, and were further analyzed for genefunctional annotation through the TAIR gene ontology (GO) anno-tation download website (http://www.arabidopsis.org/tools/bulk/go/index.jsp).

ACKNOWLEDGEMENTS

The authors thank Dr S. Rodermel for providing ftsh5 and ftsh2seeds, and Dr S. McCormick, Dr W. Briggs, Dr L. Li and Dr J. Chen fortheir critical review of the manuscript. This work was supported bygrants from the National Key Basic Research Program of China(2007CB108800), the National Natural Scientific Foundation of China(30570131), the Bairen Project of Chinese Academy of Sciences andthe Shanghai Scientific Committee (06PJ14103).

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. The genetic manipulation of endogenous GPA1 activitypartially rescued the leaf variegation of thf1.Figure S2. Identification of transgenic plants.Table S1. Among the 151 genes that are upregulated (by up totwofold) in thf1, transcripts of 49 genes are downregulated, by atleast twofold, in cGPA1/thf1.Table S2. Among 409 genes that are downregulated (by up totwofold) in thf1, transcripts of 32 genes are upregulated by at leasttwofold in cGPA1/thf1.Table S3. Primers used in this study.Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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Activation of the heterotrimeric G protein α-subunit GPA1 ...sippe.ac.cn/gh/2009 Annual Report/84.pdf · ted into the cell by heterotrimeric G proteins composed of a, b and c subunits