Heme, a Plastid-Derived Regulator of Nuclear Gene ...b Institut fu¨r Biologie/Pﬂanzenphysiologie, Humboldt Universita¨t, D-10115 Berlin, Germany To gain insight into the chloroplast-to-nucleus

Heme, a Plastid-Derived Regulator of Nuclear GeneExpression in Chlamydomonas W

Erika D. von Gromoff,a Ali Alawady,b Linda Meinecke,a Bernhard Grimm,b and Christoph F. Becka,1

a Fakultät für Biologie, Institut für Biologie III, Universität Freiburg, D-79104 Freiburg, Germanyb Institut für Biologie/Pflanzenphysiologie, Humboldt Universität, D-10115 Berlin, Germany

To gain insight into the chloroplast-to-nucleus signaling role of tetrapyrroles, Chlamydomonas reinhardtii mutants in the

Mg-chelatase that catalyzes the insertion of magnesium into protoporphyrin IX were isolated and characterized. The four

mutants lack chlorophyll and show reduced levels of Mg-tetrapyrroles but increased levels of soluble heme. In the mutants,

light induction of HSP70A was preserved, although Mg-protoporphyrin IX has been implicated in this induction. In wild-type

cells, a shift from dark to light resulted in a transient reduction in heme levels, while the levels of Mg-protoporphyrin IX, its

methyl ester, and protoporphyrin IX increased. Hemin feeding to cultures in the dark activated HSP70A. This induction was

mediated by the same plastid response element (PRE) in the HSP70A promoter that has been shown to mediate induction by

Mg-protoporphyrin IX and light. Other nuclear genes that harbor a PRE in their promoters also were inducible by hemin

feeding. Extended incubation with hemin abrogated the competence to induce HSP70A by light or Mg-protoporphyrin IX,

indicating that these signals converge on the same pathway. We propose that Mg-protoporphyrin IX and heme may serve as

plastid signals that regulate the expression of nuclear genes.

INTRODUCTION

During evolution of the chloroplast from a cyanobacterial pro-

genitor, most of the genes from the endosymbionts were trans-

ferred to the nuclear genomes of their hosts (Martin et al., 2002;

Leister, 2003). In the case of Chlamydomonas reinhardtii, the

chloroplast retained 72 genes (Maul et al., 2002). However, chlo-

roplasts are estimated to harbor ;3000 proteins (Abdallah et al.,2000), the vast majority being encoded by the nucleus, translated

in the cytosol, and subsequently transported into chloroplasts.

Some of these proteins regulate essential steps of gene expres-

sion within the chloroplast (Goldschmidt-Clermont, 1998; Leon

et al., 1998). In the course of evolution, the plastids acquired new

properties, such as the ability to import proteins and to generate

retrograde signals that in turn regulate the expression of nuclear

genes. Research in recent years has provided evidence for

multiple retrograde signaling pathways by which the chloroplast

regulates a subset of nuclear genes, most of which encode

chloroplast-localized proteins involved in photosynthesis. Among

the pathways studied in some detail, one is dependent on

products of plastid protein synthesis, for another the signal is

singlet oxygen, a third employs chloroplast-generated hydrogen

peroxide, a fourth is controlled by the redox state of the photo-

synthetic electron transport chain, and a fifth involves anabolic

intermediates and possibly proteins of the tetrapyrrole biosyn-

thesis pathway (reviewed in Rodermel, 2001; Gray et al., 2003;

Pfannschmidt, 2003; Beck, 2005; Nott et al., 2006).

One retrograde signaling pathway for which many data have

accumulated involves components of tetrapyrrole biosynthesis

(reviewed in Strand, 2004; Beck, 2005; Beck and Grimm, 2006;

Nott et al., 2006). In all plants, the steps of tetrapyrrole biosyn-

thesis up to protoporphyrinogen IX (Protogen) occur only within

the chloroplast but are catalyzed by nuclear-encoded enzymes

(Beale, 1999; Papenbrock and Grimm, 2001). In higher plants, a

fraction of Protogen leaves the chloroplast by unknown routes

and enters the mitochondria where it serves as precursor of

heme. Protogen in the chloroplast serves as precursor of both

heme and chlorophyll (Papenbrock and Grimm, 2001). In Chla-

mydomonas, in contrast with higher plants, the enzymes for the

conversion of Protogen into protoporphyrin IX (Proto) and the

subsequent insertion of Fe2þ into Proto by ferrochelatase have

recently been suggested to be exclusively located within the

plastids (van Lis et al., 2005). In this alga, only single genes exist

for Protogen oxidase and ferrochelatase whose products have

been shown by immunolocalization to localize to chloroplasts

(van Lis et al., 2005). Thus, in Chlamydomonas, all heme require-

ments of the cell appear to be met by the chloroplast, implying a

constant flux of heme to all cellular compartments.

Insertion of magnesium into Proto forms Mg-protoporphyrin IX

(MgProto) and is catalyzed by Mg-chelatase (CHL), a membrane

interacting enzyme composed of three different subunits: CHLD,

CHLH, and CHLI (Willows et al., 1996). In norflurazon-treated

Arabidopsis thaliana, an increased accumulation of MgProto has

been shown to lead to the repression of a nuclear light harvesting

chlorophyll a/b binding protein (Lhcb) gene (Strand et al., 2003).

Also, in cress (Lapidium sativum) and barley (Hordeum vulgare),

the accumulation of Mg-tetrapyrroles correlated with a reduced

expression of nuclear photosynthetic genes (Oster et al., 1996;

La Rocca et al., 2001). Furthermore, a genetic screen based on

the use of norflurazon has allowed the identification of a series of

Arabidopsis genome uncoupled (gun) mutants that are modified

1 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Christoph F. Beck([email protected]).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.107.054650

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in chloroplast-to-nucleus signaling (Susek et al., 1993). Four of

the mutants (gun2-5) were shown to have lesions in genes

involved in tetrapyrrole synthesis and/or degradation. In gun5,

which harbors a partially functional mutation of the CHLH gene,

the steady state level of MgProto in norflurazon-treated plants

was reduced relative to the wild type, correlating with a reduced

repression of Lhcb (Mochizuki et al., 2001; Strand et al., 2003).

The incubation of leaf protoplasts with MgProto, but not with

porphobilinogen, Proto, or heme, repressed Lhcb expression,

suggesting a role of MgProto in gene repression. Transgenic

tobacco (Nicotiana tabacum) plants that exhibit either over- or

underexpression of CHLM, the gene encoding MgProto methyl

transferase, contained either decreased or increased pool levels

of MgProto, and this correlated with either elevated or reduced

expression of Lhcb and HemA (glutamyl tRNA reductase [GluTR]),

GSA (glutamate-1-semialdehyde aminotransferase), and CHLH

(the H subunit of Mg-chelatase) (Alawady and Grimm, 2005). Also,

confirming previous findings, a recently described Arabidopsis

mutant that accumulates MgProto due to a mutation in CHLM

showed repression of Lhcb also in the absence of norflurazon

(Pontier et al., 2007).

The regulation of nuclear genes by chloroplast-derived signals

has been examined by studying the induction of the nuclear gene

for the chaperone HSP70 in the green alga Chlamydomonas.

Feeding of MgProto or its methylester (MgProtoMe) to Chlamy-

domonas induces HSP70A (Kropat et al., 1997). Recently, a

cis-acting plastid response element (PRE) with qualities of an

enhancer was identified in the HSP70A promoter. PRE mediates

MgProto induction of HSP70A; other genes that harbor PRE in

their promoters were likewise induced by MgProto feeding

(Vasileuskaya et al., 2004; von Gromoff et al., 2006). In Chlamy-

domonas, MgProto appears to mediate the light induction of

HSP70A seen after a shift of cultures from dark to light. Thus, a

CHLH mutant, which does not produce MgProto due to a mu-

tation in the gene for the H-subunit of Mg-chelatase prevented

the light induction of HSP70A, as did conditions abrogating the

accumulation of MgProto in wild-type strains, such as the ad-

dition of cycloheximide or cells that are partially differentiated

toward gametes (pregametes). However, in all cases, feeding of

MgProto to cultures growing in the dark resulted in HSP70A

induction, supporting a role of MgProto as a messenger in the

signaling pathway by which light induces the HSP70A chaperone

gene (Kropat et al., 1997, 2000). For this induction, the Mg-

tetrapyrroles may be made available to the cytosol and the

nucleus in a light-dependent manner (Kropat et al., 2000; Beck,

2005). MgProto in the cytosol after Norflurazon treatment has

recently been observed in Arabidopsis using confocal laser

scanning spectroscopy (Ankele et al., 2007). The observed light

induction of HSP70A via tetrapyrroles is supported by an analysis

of mutations in PRE: mutant PREs abolished induction of a fused

reporter gene by light and MgProto (von Gromoff et al., 2006).

However, a unified view on the regulatory role of tetrapyrrole

biosynthesis is not yet in sight. An alternative hypothesis has

been proposed that variations in Mg-porphyrin levels are sensed

at the sites of Mg-porphyrin synthesis by an enzyme complex and

subsequently transmitted to the cytosol/nucleus (Alawady and

Grimm, 2005; Beck and Grimm, 2006).

A new set of mutants affecting genes of CHL subunits and thus

causing diminished formation of MgProto allowed us to take a

new look at the role of tetrapyrroles in chloroplast-to-nucleus

communication. These mutants accumulate heme, which could

be shown to control the same set of genes previously identified as

inducible by MgProto. The signaling pathways of both tetrapyr-

roles converge at the same cis-acting element.

RESULTS

Identification of Mutants Defective in CHL Subunits D and H

To examine the role of MgProto in chloroplast-to-nucleus com-

munication, we first isolated mutants in the subunits of CHL. To

isolate mutants, we screened UV-mutagenized lines for chloro-

phyll deficiency. Four mutants, which were generated by UV

mutagenesis, lacked chlorophyll and showed the formation of

yellow-brown colonies. Based on the initial determination of

Proto levels, these mutants were selected as potentially defec-

tive in CHL activity. CHL is a heterotrimeric complex composed

of subunits named D, H, and I (reviewed in Walker and Willows,

1997). Analysis of the Chlamydomonas genome sequence re-

vealed a single gene for subunit D, one gene and a presumed

pseudogene for subunit H, and two genes for subunit I (Lohr

et al., 2005). To identify the mutant loci, we first identified BAC

clones containing the functional genes for the various CHL sub-

units using gene-specific probes. DNA of these BAC clones was

employed in complementation assays: the BAC DNA was used

to transform each mutant line; clones that could complement a

mutant produced colonies capable of autotrophic growth. Two of

the four mutants were complemented by BAC clones containing

the CHLD gene (mutants named chlD-1 and chlD-2), and two

mutants were complemented by BAC clones with CHLH (mu-

tants named chlH-1 and chlH-2) (Table 1). These data from

complementation tests with BAC clones were subsequently

Table 1. Complementation of Chlorophyll-Deficient Mutants with BAC Clones That Harbor the CHL Genes Indicated

No. of Photoautotrophic Clones Observed after Transformation Using BAC Clones with Genesa

Mutants Tested CHLH CHLI1 CHLI2 CHLD No DNA

CF194 chlH-1 16 0 0 0 1

CF215 chlD-1 0 0 0 16 0

CF225 chlD-2 0 1 0 134 2

CF227 chlH-2 14 0 0 0 0

a Transformations were performed as described in Methods.

Heme Regulates Nuclear Genes 553

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confirmed by transformation with the subcloned genes (data not

shown).

Characterization of Defects in Mutants chlD-1 and chlD-2

Chlamydomonas mutants defective in CHLH have previously

been characterized (Chekounova et al., 2001). We therefore

focused our mutant characterization on chlD-1 and chlD-2. The

structure of the Chlamydomonas CHLD gene, as deduced from

combined genomic and EST sequence data (http://genome.jgi-

psf.org/Chlre3/Chlre3.home.html), is presented in Figure 1A. The

mutations in the gene were identified by sequencing of PCR

fragments generated with genomic DNA of the mutants. In the

chlD-2 mutant, a transversion mutation was discovered in exon

12 (a C-to-A change), which resulted in the generation of a stop

codon (Figure 1A). Analysis of mutant chlD-1 revealed a G-to-T

transversion at a highly conserved position at the 59 intron side ofthe border between exon 11 and intron 11 (Figure 1A). To

elucidate the effect of this mutation, the mRNA in this mutant

was analyzed by RT-PCR employing primers homologous to

exon 10 and exon 12. RT-PCR of mRNA from chlD-1 yielded two

products: one slightly smaller than the wild-type product (610

versus 630 bp) and the other distinctly larger (798 bp).

The sequence of the short product revealed that removal of

intron 11 had taken place. But, in addition, 20 nucleotides from

the 39 end of exon 11 were missing (Figure 1B). The splicingmachinery apparently recognized an alternative 59 splice site inthe sequence GGT//GTA located within exon 11, 20 nucleotides

upstream from the wild-type splice site. Consensus sequences

for 59 and 39 splice sites in Chlamydomonas are (C/A)(A/C)GA//GT(G/A) (Silflow, 1998). At the exon side, specificity appears to

be relaxed since all nucleotides have been observed at these

three positions (Silflow, 1998). Splicing at this alternative position

results in a shift of the reading frame that ends, after codons for

40 amino acids, with a stop codon. In the case of the longer RT-

PCR product, intron 11 was not removed. In this case, a stop

codon occurs after 12 intron-derived codons (Figure 1B).

Proto and Heme Accumulate in CHL Mutants

While chlorophyll in all four mutants was very low or not detect-

able, the concentration of noncovalently bound heme in the

mutants was elevated twofold to fivefold compared with the wild

type, and the substrate of CHL, Proto, accumulated to levels

more than ninefold higher than in the wild type (Table 2). The

levels of MgProto were distinctly lower in all mutants. Mg-

protoporphyrin IX monomethyl-ester (MgProtoMe) levels also

were reduced (Table 2). The elevated heme levels suggest that

the impairment of chlorophyll synthesis in CHL-defective mu-

tants, resulting in an accumulation of Proto, enhances heme

synthesis.

Regulatory Properties of the CHL Mutants

Since subunits of CHL have been suggested to participate in the

regulation of nuclear genes (Papenbrock et al., 2000a, 2000b;

Mochizuki et al., 2001), we first tested whether the light regulation

of all four functional CHL genes was affected in the chlD and chlH

mutants. In the parental strain, all four genes exhibited a light-

induced accumulation of their transcripts (Figure 2). This induc-

tion was dependent on cytoplasmic protein synthesis since

presence of the inhibitor cycloheximide prevented this light-

induced mRNA accumulation (see Supplemental Figure 1 online).

By contrast, inhibition of plastid protein synthesis by chloram-

phenicol had no effect (see Supplemental Figure 1 online). The

chlD and chlH mutants showed accumulation of their mRNAs

that were similar to those of the wild type (Figure 2). Analysis of

the expression patterns of the various CHL genes in the chlD and

chlH mutants revealed no significant changes in light-induced

mRNA accumulation (Figure 2). Although the kinetics of mRNA

accumulation exhibited some strain-dependent variations, an

increase in mRNA levels was observed for all mutants in inde-

pendent experiments, indicating that light induction is not

blocked by the accumulation of Proto or reduced levels of Mg-

tetrapyrroles in the first 4 h of low-level irradiation.

Figure 1. Structure of the CHLD Gene and Alterations Caused by the

Mutations chlD-1 and chlD-2.

(A) Gene CHLD with 12 exons. Dark-gray boxes indicate deduced

protein coding exons, open boxes indicate introns, and black bars

indicate 59 and 39 untranslated regions. The positions and sequence

alterations of mutations chlD-1 and chlD-2 are indicated.

(B) RT-PCR sequences covering the splice site alteration caused by

mutation chlD-1 document the changes at the mRNA level. Sequences

from the wild type and the two chlD-1 mutant products are shown.

Vertical arrows indicate the end of exon 11. The altered base at the

junction of exon 11 and intron 11 is indicated by a bold letter in the long

RT-PCR fragment. Deduced amino acid sequences that differ from the

wild type are shown in bold and italics. Asterisks indicate stop codons.

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We analyzed the expression of HSP70A in response to light

and observed an induction in the wild-type strain and in the four

mutants defective in CHL (Figure 3). A reduction of approximately

twofold in induced HSP70A mRNA levels compared with the wild

type was observed in the chlD mutants. Also, progeny of chlD

mutants from crosses with the wild type showed this reduction,

indicating that this reduction is apparently not due to second-site

mutations in the original mutants. We included a previously char-

acterized CHLH-defective mutant, brs-1 (though a clone of dif-

ferent origin than the one originally used by Kropat et al., 1997),

and observed light induction of the heat shock gene. Using the

four newly isolated mutants, these data document that a block in

the synthesis of Mg-tetrapyrroles does not prevent the induction

of HSP70A by light.

A Role for Heme as an Inducer of HSP70A

We previously postulated an essential role for MgProto and/or

MgProtoMe in the light induction of HSP70A (Kropat et al., 1997,

2000; Beck, 2005). Such a model was supported by the discov-

ery of sequence elements within the HSP70A promoter called

PREs that mediate HSP70A induction in response to MgProto

feeding (von Gromoff et al., 2006). Since in the CHL mutants

heme was shown to accumulate (Table 2) and heme previously

has been identified as a regulator of the nuclear gene HEMA

(Vasileuskaya et al., 2005), we speculated that heme also plays a

role in HSP70A expression. This was tested by feeding of hemin

(which harbors a Fe3þ atom, conferring higher stability in solution

than the Fe2þ contained in heme) to cultures in the dark. The data

shown in Figure 4A reveal that hemin feeding in the dark induced

HSP70A mRNA accumulation. The kinetics of mRNA accumu-

lation upon hemin feeding were delayed compared with MgProto

feeding, but the degree of induction with 9 mM hemin was similar

to that seen after MgProto feeding (9 mM) or a shift from dark to

light (DLS). We conclude that heme, just like MgProto, may

activate HSP70A, while Proto and protochlorophyllide, as shown

previously (Kropat et al., 1997), have no inducing effect.

To verify and extend these studies, we analyzed the induction

of HSP70A by various concentrations of hemin. As shown in

Figure 4B, HSP70A mRNA levels rose with an increase in the

hemin concentration applied to cell cultures in the dark. Maximal

induction was observed upon feeding of 16 mM hemin. In sub-

sequent experiments, a final concentration of 9 mM hemin was

employed.

Changes in Tetrapyrrole Levels after a Shift from

Dark to Light

Exposure of dark-adapted wild-type cells to light resulted in an

immediate rise (within

drop in pool levels in the 20 min samples, followed by a second

increase. Such fluctuations, also observed by others (Pöpperl

et al., 1998), may reflect a fine-tuning process among the enzy-

matic activities of CHL and downstream enzymes affecting the

regulatory equilibrium after a sudden shift of cultures from dark to

light. Upon extended exposure of cultures to light (i.e., >60 min),

the increase of tetrapyrroles stops and reduced pool levels were

observed for Proto and MgProto (Figure 5). A similar rapid and

transient, light-induced increase in pool sizes of MgProto and

MgProtoMe has also been observed in higher plants grown in

light/dark cycles (Pöpperl et al., 1998; Papenbrock et al., 1999).

By contrast, levels of total noncovalently bound heme, deter-

mined from the same cell cultures, exhibited an approximately

twofold transient reduction in pool levels after a delay of ;10 min(Figure 5). This decrease may possibly be accounted for by an

enhanced demand of heme for assembly into proteins. Alterna-

tively, the decrease in heme levels may reflect a channeling of

Proto into the Mg-branch for chlorophyll biosynthesis. A prefer-

ential channeling may be explained by an activation of CHL under

these conditions. Indeed, an increase in CHL activity upon shift of

cells from dark to light was observed (Figure 6). This increase

showed some fluctuations (Figure 6). An elevated CHL activity

seen 5 min after start of illumination was followed by a decrease

seen at 10 min, and this again was followed by an increase.

These initial fluctuations in CHL activity are mirrored by changes

in MgProto and MgProtoMe pool levels right after start of

illumination (Figure 5). In the latter, an initial peak at 10 min was

followed by lowered pool levels at 20 min. However, whether

there is a strict interdependence between the fluctuations at the

level of enzyme activity and pool levels remains to be investi-

gated. The rapid change in CHL activity precludes an enhanced

de novo synthesis of the enzyme as an explanation for the

increase in activity. Rather, the data support a light activation of

the enzyme at the posttranslational level, possibly via thiore-

doxins, which have been shown to interact with CHL. A stimu-

lation of the enzyme by reduction was observed in isolated

chloroplasts (Balmer et al., 2003; Ikegami et al., 2007).

We also analyzed the tetrapyrrole pools of the mutants upon

shift from dark to light. No significant changes were observed

(i.e., heme and Proto pools remained high; Table 2), while the

Mg-tetrapyrrole pools remained at a low level (see Supplemental

Figure 2 online).

Is the Same cis Sequence Element Involved in the Induction

of HSP70A by Heme and MgProto?

The data presented above suggest that heme and the magne-

sium containing porphyrins act through a similar or even identical

signaling pathway for the induction of HSP70A. To study this is-

sue in more detail, we tested whether heme induction is possibly

mediated via the same regulatory regions within the HSP70A

promoter. For these analyses, we used constructs in which the

HSP70A promoter (PA), fused to the RBCS2 promoter (PR), was

located upstream of a reporter gene (HSP70B-TAG) (Figure 7A).

In transformants with such constructs, induction of the reporter

gene by light or MgProto has previously been shown to occur via

activation of the PR promoter by enhancer elements within PA(von Gromoff et al., 2006). Thirty transformants with each of the

constructs shown in Figure 7A were analyzed for expression of

the reporter gene after shift from dark to light (DLS) or after

feeding (in the dark) of MgProto or hemin (9 mM final concentra-

tion each). In agreement with previous data, MgProto activation

Figure 3. Light Induction of HSP70A in the Wild Type and Mutants Defective in the D and H Subunits of CHL.

Cultures grown in the dark at time 0 were shifted into dim light (10 mE m�2 s�1), and samples for RNA isolation were taken at the time points indicated

(hours). RNA gel blot hybridizations were performed using probes specific for HSP70A and CBLP, the latter serving as a loading control. The quantitative

evaluation of the RNA gel blots in the case of the wild type and mutants chlH-1, chlH-2, brs-1, comprised four independent experiments. In the case of

chlD-1 and chlD-2, the data from three independent experiments with the original mutant strains and of four mutant progenies from crosses with the wild

type were combined. The average levels of mRNA accumulation relative to the dark control (corrected for differences in loading) 6 SE are given.

556 The Plant Cell

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of PR was seen in transformants even with the shortest HSP70A

promoter fragment (Figure 7A). This fragment, named regulatory

region I, has been shown to harbor an enhancer element that may

confer inducibility by MgProto to heterologous promoters (von

Gromoff et al., 2006). Clearly, heme activation also is mediated

via regulatory region I (Figure 7A).

We next tested whether the element that mediates induction by

MgProto within regulatory region I also is involved in the activa-

tion by hemin. Within regulatory region I, the PRE sequence motif

GCGACNAN15TA was shown to confer MgProto responsiveness

(von Gromoff et al., 2006). Two mutant constructs that lacked

sequences either from the 59 or the 39 ends of regulatory region I(mutations 1 and 2 in Figure 7B), and thus were defective in PRE,

were tested for transcriptional activation of the reporter gene.

Transformants with these constructs did not show a response to

either hemin or MgProto. By contrast, transformants with the

wild-type construct or a deletion construct lacking five internal

base pairs but maintaining the PRE motif (mutation 3) were

inducible by both tetrapyrroles (Figure 7B). From these results we

conclude that heme induction appears to be mediated via PREs,

suggesting also that it may employ the same or similar protein

components as MgProto to activate HSP70A.

Hemin Activates Other MgProto-Responsive Genes

We have previously identified a group of nuclear genes that were

inducible by MgProto feeding (Vasileuskaya et al., 2004; von

Gromoff et al., 2006). All of these genes harbor in their promoter

regions the cis-acting PRE motif (von Gromoff et al., 2006). The

response of these genes to MgProto in wild-type strain CC-124

was confirmed (Figure 8). All five genes also responded to hemin

feeding, although the kinetics and the degree of mRNA accumu-

lation showed some variation compared with MgProto feeding,

suggesting the involvement of additional factors in the expres-

sion of these genes. The effect of the cell wall on the kinetics of

hemin induction was assessed using the cell wall–deficient mu-

tant CW15. The response of the genes in this mutant to hemin

appears to be more rapid for all genes (Figure 8), suggesting that

the cell wall represents some barrier for hemin uptake. These

results show that in Chlamydomonas a number of genes exists

that responds to two different tetrapyrroles, heme, and MgProto.

These data support the concept of an overlapping role for heme

and MgProto that, as organelle-derived molecules, appear to

play a regulatory role in transcription within the nucleus.

Assay of Other Metalated Porphyrins

To test whether only specific tetrapyrroles play a role in gene reg-

ulation, we tested the effect of three additional metalated por-

phyrins. In contrast with the Mg- and Fe-derivatives, neither Zn,

Co, nor Mn-protoporphyrin IX, when fed to cultures in the dark,

resulted in induction of HSP70A or HEMA (Figure 9A). Also, Proto

applied at higher concentrations had no effect on the expression

of the genes tested (Figure 9B). We previously showed that Proto

fed to cultures in the dark results in the accumulation of MgProto.

This MgProto, produced by chloroplast-localized enzymes, ap-

parently is not accessible to cytosol/nucleus in dark-grown cul-

tures (Kropat et al., 2000). These results suggest a high degree of

Figure 4. Changes in HSP70A mRNA Levels after Treatment of Cultures

in the Dark with Hemin.

(A) RNA accumulation in wild-type cultures that, after an incubation for

16 h in the dark (D), were treated with hemin (9 mM), MgProto (9 mM), or

shifted to light (60 mE m�2 s�1, DLS) for the given number of hours.

Samples for RNA isolation were taken at the times indicated and RNA gel

blot hybridizations were performed using probes specific for HSP70A

and CBLP, the latter serving as a loading control.

(B) Dependence of HSP70A mRNA accumulation on hemin concentration.

Cells were harvested, and RNA was extracted 2 h after addition of hemin at

the concentrations indicated to wild-type cultures incubated in the dark.

The HSP70A mRNA accumulation observed in a representative experiment

is given relative to that of the culture that did not receive hemin.


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specificity in the interaction of Mg- and Fe-porphyrins with down-

stream signaling components.

Extended Incubation with Hemin Results in Turning off the

Downstream Tetrapyrrole Signaling Pathway

The incubation of dark-grown cells with hemin resulted in a

transient HSP70A mRNA accumulation. Twelve hours after the

addition of hemin, HSP70A mRNA levels were about the same as

prior to treatment (Figure 10), although hemin was still present at

;50% of the initial concentration (data not shown). When such ahemin-treated culture was exposed to light (DLS), at most a very

weak accumulation of HSP70A mRNA was observed (Figure 10).

For an explanation we considered the possibility that hemin still

present in the culture medium may have prevented a light-

induced accumulation of the Mg-tetrapyrroles (e.g., via feedback

inhibition of GluTR, the first enzyme specific for tetrapyrrole bio-

synthesis). Such an accumulation of Mg-tetrapyrroles has pre-

viously been shown to be essential for the HSP70A activation

that follows a shift from dark to light (Kropat et al., 2000). An

increase in Proto, MgProto, and MgProtoMe pool levels indeed

was not observed when cultures treated with hemin for 12 h in the

dark were exposed to light (see Supplemental Figure 3 online).

The defect in light induction in these cells appears to involve

additional factors. To test whether downstream signaling com-

ponents may have been affected by hemin treatment, the cul-

tures were fed with MgProto after 12 h with hemin. No induction

Figure 6. Changes in CHL Activity after a Shift of Cultures from Dark to

Light.

Activity of CHL was determined with cultures of wild-type strain 4Aþgrown in TAP medium to a density of 3 to 5 3 106 cells/mL. Exponentially

growing cultures after an incubation in dark for 16 to 18 h were

transferred into light (50 mE m�2 s�1) at time 0. The cells were harvested

at the time points indicated and disrupted by sonication at 48C. The

enzyme assays were performed as described in Methods. Each time

point represents the average of data from two independent experiments.

In each of these experiments, every time point represents a kinetic

analysis of MgProto formation from Proto (for details, see Methods).

Figure 5. Changes in Tetrapyrrole Pool Levels in the Wild Type after a

Shift from Dark to Light.

Cultures of the wild type were grown in TAP medium. Prior to dark-to-

light shift, exponentially growing cultures were incubated in the dark for

16 to 18 h. At time 0, the cultures were shifted into the light (50 mE m�2

s�1) and samples were taken at the times indicated. For heme determi-

nations, 250 mL of cells (4 3 106 cells per mL) were chilled on ice and

harvested by centrifugation at 48C. For determination of the concentra-

tion of the other tetrapyrroles, 100 mL of cells (4 3 106 cells per mL) were

chilled on ice and harvested. The harvested cells quickly were frozen in

liquid nitrogen and stored at �808C. The samples were processed forpool determinations as outlined in Methods. The average pool levels of

four to six experiments 6 SE are given.

558 The Plant Cell

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was observed (Figure 10), suggesting that either the downstream

signaling pathway has been turned off or, potentially, hemin

treatment had activated nucleases that rapidly degrade HSP70A

mRNA. To address this latter point, heat shock treatment was

performed with the cells incubated with hemin for 12 h. This

treatment elicited an initial HSP70A mRNA accumulation similar

to that observed upon the application of heat shock to hemin-

free cultures (von Gromoff et al., 1989). We conclude that hemin

treatment apparently had not impaired the potential of cells to

accumulate HSP70A mRNA (Figure 10).

Figure 7. Characterization of HSP70A Promoter Elements That Confer Inducibility by Hemin on the RBCS2 Promoter.

(A) Expression of the HSP70B-TAG reporter gene with the very weakly expressed Chlamydomonas RBCS2 promoter (PR) after upstream insertion of

various HSP70A promoter sequences (von Gromoff et al., 2006). The open bar represents HSP70A promoter (PA) sequences. The RBCS2 promoter (PR)

is represented by a black bar, and the HSP70B reporter gene is indicated by a hatched bar. Transformants harboring the various constructs were

generated using strain 325 as described previously (von Gromoff et al., 2006). They were either kept in the dark (CD) for 16 to 20 h, exposed to light for

1 h after the dark period (DLS), treated with MgProto (9 mM) in the dark (MP), or treated with hemin (9 mM) in the dark (H). The reporter gene transcripts

were detected with the 199-bp TAG probe (Kropat et al., 1995). For each construct, 30 individual transformants were assayed. The number of

transformants showing induction by the various treatments is indicated. The average levels of RNA accumulation relative to the dark control (corrected

for differences in loading) 6 SE are given below these numbers. TSA1 and TSA2 designate the transcription start sites used upon heat shock and DLS or

MgProto treatment in the native HSP70A promoter, respectively. In the constructs shown, the transcription start site employed after shift from dark to

light or after MgProto treatment is that of the RBCS2 promoter PR (von Gromoff et al., 2006), which is indicated by the boxed designation DLS/MP/H.

HSE, heat shock element.

(B) Analysis of the effect of mutations in region I, the �81 to �55 PRE-containing fragment, on the activation of PR by hemin and MgProto. The basicconstruct employed is the third from top in (A). RNA dot blot analyses of 30 transformants for each construct are presented. The deletions (D) introduced

in region I are indicated. The actual nucleotide sequence located in front of PR is also shown. Shadowed in gray is the consensus sequence of the PRE

defined previously (von Gromoff et al., 2006). The dot blot signals resulting from hybridization with the TAG probe from individual transformants either

incubated in the dark, or with 9 mM MgProto, or with 9 mM hemin for 1 h were compared. The fold change in average signal intensity compared with

cultures kept in the dark 6 SE are given. The last column gives the percentage of transformants that exhibited a signal by dot blot analysis.


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DISCUSSION

Here, we report on experiments that allow a new look at the role

of tetrapyrroles in chloroplast to nucleus signaling. They are

based on four newly identified mutants with defects in CHL, the

first enzyme specific for the chlorophyll biosynthetic branch. Two

of the mutants were shown to be defective in the H subunit of the

enzyme since they could be complemented by a wild-type CHLH

gene (Table 1). Two other mutants are defective in the D subunit.

These four mutants exhibited either no or only residual levels of

chlorophyll (Table 2), indicating that the mutations blocked

chlorophyll synthesis. All four mutants accumulated Proto (Table

2). In contrast with Chlamydomonas, an accumulation of Proto

has not been observed in higher plants with deficient CHL activity

(Papenbrock et al., 2000b). However, accumulation of Proto was

seen in other Chlamydomonas mutants defective in CHLH (Wang

et al., 1974; Kropat et al., 2000; Chekounova et al., 2001).

Analysis of the expression of nuclear genes upon shift of

cultures from dark to light revealed that the four mutants did not

exhibit significant differences in the light-induced expression of

the CHL genes (Figure 2), HEMA, ALAD, or several LHC genes.

Also, basal level expression of these genes appeared not to be

affected (L. Meinecke and C.F. Beck, unpublished data).

The observation that HSP70A was light inducible in the mu-

tants (Figure 3) at first glance was surprising, since previously a

mutant defective in CHLH (brs-1) did not show light induction of

HSP70A (Kropat et al., 1997). A strain with this mutation, al-

though a different subclone, was included in our studies and here

showed light induction of HSP70A (Figure 3). The lack of light

induction in previous experiments may be explained by a sec-

ond-site mutation in the brs-1 mutant clone originally tested.

Indeed, we discovered second-site mutations that caused al-

tered regulation of nuclear genes with high frequency in a screen

of mutants defective in chlorophyll synthesis. Thus, five out of 13

Figure 8. Test of MgProto-Inducible Genes for Induction by Hemin.

Wild-type cells and cells of the cell wall–deficient mutant CW15 were

incubated in the dark for 16 h and then treated with hemin (9 mM) or

MgProto (9 mM). Samples for RNA isolation were taken at the times

indicated. RNA gel blot hybridizations were performed as described in

Methods using the gene-specific probes described either in Methods or

by Vasileuskaya et al. (2004). CBLP (von Kampen et al., 1994) served as a

loading control.

Figure 9. Test for Inducing Potential of Various Metalated Porphyrins and of Proto at Different Concentrations.

Cells of the wild type grown in TAP medium were incubated in the dark for 16 to 18 h. At time 0, the various porphyrins were added. Samples for RNA

isolation were taken at the times indicated. RNA gel blot hybridizations were performed using the probes described in Methods. CBLP served as a

loading control.

(A) The metalated porphyrins listed were added to a final concentration of 9 mM.

(B) The concentrations of Proto indicated were tested for inducing potential.

560 The Plant Cell

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chlH mutants from the initial screen for chlorophyll-deficient

mutants exhibited deregulation of HSP70A. In each case,

crosses revealed a segregation of the phenotypes of chlorophyll

deficiency and deregulated gene expression. For our studies, we

chose two of the chlH mutants that exhibited normal regulation of

HSP70A. In the two chlD mutants, a reproducible reduction in

HSP70A mRNA accumulation by a factor of about two was

observed upon dark-to-light shift compared with the wild type or

other CHL mutants (Figure 3). In this case, second-site mutations

being responsible for the reduced induction with high probability

could be ruled out since all mutant progenies from crosses of the

two strains with the wild type revealed a similar reduction (data

not shown). These data point to a complex role of individual CHL

subunits in signaling, a view supported by the observation that

Arabidopsis mutants with lesions in the CHLI gene do not exhibit

a gun phenotype upon norflurazon treatment (Mochizuki et al.,

2001).

In view of the accumulated evidence for a crucial role of

tetrapyrroles in mediating the light induction of HSP70A (Kropat

et al., 1997, 2000; von Gromoff et al., 2006), we considered an

involvement of other tetrapyrroles besides the Mg-tetrapyrroles

in this induction. Heme was an attractive candidate since this

tetrapyrrole activates HEMA, encoding GluTR, the first enzyme

specific for tetrapyrrole biosynthesis (Vasileuskaya et al., 2004).

Indeed, feeding experiments with hemin in the dark induced

HSP70A mRNA accumulation (Figure 4). This suggested that

heme may play a similar role to MgProto in signaling.

A crucial aspect of an understanding of tetrapyrrole gene

regulation was the definition of a sequence motif within the

HSP70A promoter that may activate the gene upon hemin

feeding. The previously identified motif (named PRE) that medi-

ates HSP70A induction by MgProto (von Gromoff et al., 2006)

was shown also to confer heme-responsive activation on

HSP70A since mutations in PRE that abolished induction by

MgProto also abolished activation by hemin (Figure 7). These

data provide solid evidence for a specific role of heme in the

regulation of nuclear genes. Indeed, other genes that harbor a

PRE were also induced by the feeding in the dark of MgProto or

hemin (Figure 8). A test of different metalated porphyrins re-

vealed that only MgProto and hemin induce PRE-harboring

genes (Figure 9). Previously we have shown that, besides

MgProto, MgProtoMe2 (used instead of the naturally occurring

monomethyl ester) also induced HSP70A, but protochlorophyl-

lide or chlorophyllide had no inducing potential (Kropat et al.,

1997). This and the inability of metalated porphyrins with metal

ions other than Mg and Fe to induce HSP70A (Figure 9) point to a

high degree of specificity in the interaction between the meta-

lated tetrapyrroles and their receptors. Whether a single receptor

with dual specificity or two receptors are involved remains to be

elucidated.

Salient features of our previous model were two prerequisites

for HSP70A light activation: first, a light-induced transient accu-

mulation of Mg-tetrapyrroles and, second, a light-activated

export of tetrapyrroles from the chloroplast (Kropat et al., 2000;

Beck, 2005; Beck and Grimm, 2006). Here, in view of our new

results, we want to modify and extend this model (Figure 11).

The reduction in heme levels that follows a shift of cultures

from dark to light (Figure 5) provides new information on

Figure 10. Response of HSP70A to Various Treatments after Extended

Incubation with 9 mM Hemin.

Cultures grown in the dark for 16 h were treated with 9 mM hemin (H) in the

dark and incubation was continued for 12h (top panel). After this incubation,

an aliquot of the culture was irradiated (60 mE m�2 s�1) for up to 2 h (DLS,

second panel from top). To another aliquot, MgProto (MP) was added to a

final concentration of 9 mM and incubation in the dark was continued for 2 h

(third panel from top). To a third aliquot, hemin (H) was added (the newly

added H corresponded to a concentration of 9 mM, but residual hemin was

present from the previous incubation) and incubation was continued for 2 h

in the dark (fourth panel from top). In another aliquot, the response of cells to

heat shock after 12 h incubation with hemin was tested. For this purpose the

culture was shifted from 23 to 408C in the dark for up to 2 h (fifth panel from

top). The disappearance of HSP70A mRNA and 18s,25s rRNA 2 h after heat

shock in cultures treated with hemin has been observed in independent

experiments, suggesting a destabilization of RNA after prolonged incuba-

tion at elevated temperature. A dark-to-light shift (DLS) was performed with

a culture not treated with hemin to illustrate the responsiveness of HSP70A

to light (sixth panel from top). In all cases, samples for RNA isolation were

taken at the times indicated. RNA gel blot hybridizations were performed

using specific probes for HSP70A mRNA and 18s,25s rRNA (labeled 18s

rRNA), the latter serving as a loading control. In these experiments, CBLP

could not be used since the mRNA of this gene disappeared upon extended

hemin treatment (>6 h).


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intraplastidal regulatory events that result in the transient accu-

mulation of Mg-tetrapyrroles upon dark-to-light shift. Three

different regulatory events may account for these changes. (1)

In Chlamydomonas cultures, a shift from dark to light not only

results in a transient increase in Proto and MgProto/MgProtoMe

pool levels but also in a decrease in noncovalently bound heme

(Figure 5). The lowering of heme levels in the chloroplasts after

shift to light can be expected to correlate with an at least partial

release of GluTR from feedback inhibition by heme (Vothknecht

et al., 1996), resulting in a higher flux through the tetrapyrrole

biosynthetic pathway and thus the accumulation of Proto,

MgProto, and MgProtoMe (pathway 1 in Figure 11). The de-

crease in heme levels in the presence of increasing Proto levels

indicates a preferential channeling of Proto into chlorophyll bio-

synthesis, which may be explained by the observed light activa-

tion of CHL (Figure 6). (2) A negative regulator of tetrapyrrole

biosynthesis that affects the Mg branch of tetrapyrrole biosyn-

thesis is the Flu protein. Mutants of Arabidopsis that carry a

lesion in the FLU gene are no longer able to downregulate

d-amino levulinic acid synthesis and thus accumulate proto-

chlorophyllide when grown in the dark (Meskauskiene et al.,

2001). The Flu protein has subsequently been shown to interact

with GluTR (Meskauskiene and Apel, 2002). A shift from dark to

light enables catalysis of light-dependent protochlorophyllide

reduction by protochlorophyllide oxidoreductase, and this, in the

case of higher plants (Meskauskiene et al., 2001) and Chlamy-

domonas (Pöpperl, 1996), has been shown to result in a reduc-

tion in protochlorophyllide levels. In light, GluTR may be relieved

from a negative control by the Flu protein, resulting in an

enhanced synthesis of tetrapyrroles (pathway 2 in Figure 11).

(3) There remains the possibility that early steps of tetrapyrrole

biosynthesis are subject to direct light activation (e.g., via post-

translational modification) (pathway 3 in Figure 11). Which of

these routes or combination of routes results in the increased

synthesis of tetrapyrroles observed upon illumination remains to

be elucidated.

In light of our model on the involvement of tetrapyrroles in

chloroplast retrograde signaling in Chlamydomonas, we can now

address the differences between green algae and higher plants.

The recent identification of the Arabidopsis GUN1 gene and the

analysis of mutants defective in this gene resulted in a model in

which a chloroplast-localized protein with DNA/RNA binding

activity (GUN1) generates a signal that coordinates nuclear gene

expression in response to various alterations within the plastids

(i.e., the accumulation of MgProto, inhibition of plastid gene ex-

pression, and the redox state of the photosynthetic electron

Figure 11. Model for the Activation of the HSP70A Promoter by MgProto and Heme.

A strongly abbreviated pathway for the synthesis of heme and chlorophyll, starting with Glu, is indicated. (1) Feedback inhibition of GluTR by heme;

(2) feedback regulation from the Mg-tetrapyrrole branch of the pathway mediated by the Flu protein (Goslings et al., 2004); (3) a direct activating effect

of light on the first steps of tetrapyrrole biosynthesis. Details of the model are presented in the Discussion.

562 The Plant Cell

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transfer chain) (Koussevitzky et al., 2007). Our data obtained with

the green alga Chlamydomonas reveal a number of differences:

(1) The sequence motifs that meditate the response to elevated

MgProto levels in higher plants and Chlamydomonas are not re-

lated (Strand et al., 2003; von Gromoff et al., 2006; Koussevitzky

et al., 2007). In Arabidopsis, the ABI4 transcription factor medi-

ates the response to a signal from GUN1; in Chlamydomonas, the

factor that binds PRE is still elusive. (2) Heme in Chlamydomonas

has a regulatory function in gene regulation; in Arabidopsis, no

evidence was observed for a role of heme similar to that of

MgProto (i.e., in the repression of Lhcb) (Strand et al., 2003). (3)

Within the Chlamydomonas HSP70A promoter, three different

regions have been identified that mediate activation of the gene

in response to tetrapyrroles, hydrogen peroxide, and singlet

oxygen (Shao et al., 2007). This indicates that in Chlamydomonas

different plastid signals appear not to converge in a common

pathway. (4) A gene with homology to GUN1 (i.e., encoding a

pentatricopeptide repeat protein with a mutS-related domain)

was not detected in the Chlamydomonas genome sequence

(http://genome.jgi-psf.org/Chlre3/Chlre3.home.html).

How can the light induction of HSP70A, previously postulated

to be mediated by MgProto, be explained in view of the reduced

MgProto levels in the four CHL mutants? Here, we propose that,

besides MgProto, heme may also mediate light induction. How-

ever, this function of heme may be restricted to a special set of

conditions (e.g., when Mg-tetrapyrrole levels are low and heme

levels are elevated). The heme levels in the mutants are twofold

to fivefold higher than in the wild type (Table 2), and they do not

decrease upon shift of mutant cultures from dark to light (see

Supplemental Figure 2 online). Since the mutants retain HSP70A

activation in response to light, we postulate that heme in a light-

dependent step is made available to a downstream signaling

pathway in the cytosol/nucleus (see below) (Figure 11).

The presence of a signaling pathway for heme, including a

defined promoter element by which heme may modulate gene

expression, is viewed as a strong indicator for a regulatory role of

heme in Chlamydomonas. We envision that, besides a role in light

induction of PRE-harboring genes, heme may have regulatory

functions under physiological conditions when heme levels are

elevated but MgProto is not available or strongly reduced. Such

conditions in plants exist in non-green tissue but also have been

documented for tobacco grown under a 12-h-light/12-h-dark

cycle where pool levels of various tetrapyrroles were shown to

change in opposite directions. Toward the end of the light period,

pools of Proto, MgProto, and MgProtoMe were shown to drop

approximately fivefold, while heme levels increased ;1.5-fold(Papenbrock et al., 1999).

In Chlamydomonas, the chloroplast has been suggested to be

the only site of heme synthesis (van Lis et al., 2005), and heme,

also in the dark, will constantly be required to serve as prosthetic

group of proteins in the cytosol and the mitochondria. Thus, a low

level of heme must constantly exit from the chloroplast. Heme

efflux from chloroplasts has indeed been observed (Thomas and

Weinstein, 1990). We can envision that the Km of the heme

binding factor(s) involved in signaling in the cytosol is significantly

higher than that of reactions involved in the insertion of heme as a

prosthetic group. The signaling factors thus only may become

activated when the heme concentration in the cytosol/nucleus is

raised (e.g., after an active, light-driven export of heme from the

chloroplast). This hypothesis may be tested once heme binding

factors have been identified.

Information about the downstream signaling pathway was

gained by experiments in which cultures were incubated with

hemin for 12 h (Figure 10). This pathway was shown to share a

genuine property of most signaling pathways (i.e., it is inactivated

upon prolonged stimulation). In cells treated with hemin for 12 h,

HSP70A light induction was essentially abolished (Figure 10).

Since the addition of MgProto in the dark also did not elicit an

induction of HSP70A, we may conclude that shared components

of the downstream signaling pathway are blocked. The lack of

HSP70A induction by either MgProto or hemin feeding suggests

that either the receptor(s) for these tetrapyrroles or downstream

components got inactivated. Indeed, the employment of the

same cis-acting PRE element of the HSP70A promoter for the

activation by both MgProto and heme (Figure 7) may point to a

sharing of transcription factors by the two tetrapyrroles.

These studies provide evidence for a regulatory role of heme in

gene expression in Chlamydomonas. Whether this tetrapyrrole

also is active in gene regulation in higher plants remains to be

elucidated. In yeast and animal systems, a role of heme in

regulation of gene expression is well established (reviewed in

Mense and Zhang, 2006). Thus, in mammals, heme has recently

been demonstrated to be an amplifier of the inflammatory

response via specific activation of toll-like receptor 4 (Figueiredo

et al., 2007). The details of the signaling pathways in Chlamydo-

monas can now be addressed, and this may provide clues to a

regulatory role of heme in higher plants.

METHODS

Algal Strains and Culture Conditions

Chlamydomonas reinhardtii strain 325 (cw15, arg7-8, mtþ) was kindlyprovided by R. Matagne (University of Liège, Belgium). Wild-type strain

CC-124 (mt�) and the cell wall–deficient mutant CW15 were obtainedfrom the Chlamydomonas Culture Collection at Duke University. Wild-

type strain 4Aþ (mtþ) was provided by J.-D. Rochaix (University ofGeneva). The mutants were generated by M. Kobayashi in the 4Aþ strainby UV mutagenesis applying between 30 and 60 mJ cm�2. After muta-

genesis, the cells were plated and incubated in the dark. Mutant clones

were recognized by their altered pigmentation. If not otherwise noted,

strains were grown photomixotrophically in Tris-acetate phosphate (TAP)

medium (Harris, 1989) on a rotatory shaker at 238C under continuous

irradiation with white light (;60 mE m�2 s�1) provided by fluorescenttubes (Osram L36W/25). Mutant stocks were maintained on TAP agar

medium in the dark. Light induction and heat shock were performed

according to Kropat et al. (1995) and von Gromoff et al. (1989), respec-

tively. For Arg-requiring strains, the medium was supplemented with Arg

(final concentration 100 mg/mL).

Nuclear Transformation of Chlamydomonas

Chlamydomonas nuclear transformation was performed using the glass

bead method (Kindle, 1990), modified as described previously (Kropat

et al., 2000). For the transformation of 108 cells, 100 ng of plasmid DNA or

1 mg of BAC DNA were used. Immediately after vortexing with glass

beads, cells were spread onto freshly prepared TAP-agar (1%) plates.

Plates were incubated at 238C in the light (;60 mE m�2 s�1), and


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transformants were collected and analyzed after 1 week. For promoter

activity tests, strain 325 was transformed as described by von Gromoff

et al. (2006). The constructs (all containing the ARG7 gene) were linear-

ized by restriction either upstream from HSP70A promoter sequences or

downstream from the reporter gene.

RNA Gel Blot Analysis

Total RNA was isolated from 10 mL cultures grown to 2 to 4 3 106 cells per

mL. The procedures employed for RNA extraction, separation on agarose

gels, blotting, and hybridization were as described previously (von

Gromoff et al., 1989) except that the nylon membranes used were

Hybond-N (Amersham). The probes used for hybridization were as

follows: HSP70A (von Gromoff et al., 1989), CHLH 1 (Chekounova

et al., 2001), CHLI 1 (a SpeI-XhoI cDNA fragment of ;1.5 kb from ESTclone AV642320), CHLI 2 (a SpeI-XhoI fragment of ;1.8 kb from ESTclone AV623288), CHLD (a 680-bp cDNA fragment), and HEMA (a 560-bp

cDNA fragment), kindly provided by S. Beale. The EST clones were

obtained from the Japanese collection (Asamizu et al., 1999). Either

CBLP, which encodes a Gb-like polypeptide (von Kampen et al., 1994), or

a clone (Ba235) harboring the 18s and 25s rRNA genes (Marco and

Rochaix, 1980) was used as loading controls. Probes were labeled with

50 mCi [a-32P]dCTP (Amersham) according to the random priming pro-

tocol (Feinberg and Vogelstein, 1983). Hybridizations were performed as

described previously (Wegener and Beck, 1991). After hybridization, the

membranes were washed twice in 23 SSC for 5 min at room temperature,

once in 23 SSC, 1% SDS for 30 min at 658C, and once in 0.13 SSC, 1%

SDS for 30 min at 658C. RNA gel blots were screened by exposing

membranes to BAS-MP imaging plates (Fuji). They were evaluated by a

phosphor imager (Bio-Rad Laboratories). For the quantitative determi-

nation of changes in mRNA concentrations, all clones that exhibited a

response were evaluated using the Quantity One 4, 5, 1 program from

Bio-Rad Laboratories.

RNA Dot Blot Analysis

Ten micrograms of total RNA from each sample was applied to Hybond-N

nylon membranes using a dot blot apparatus. The membranes were dried

and RNA fixed to the membranes by baking for 2 h at 808C. For

hybridization, washing of the membranes, and their quantitative evalua-

tion, we used the same conditions/programs as for RNA gel blot analyses.

Isolation of Genes for CHL

BAC clones containing genes CHLH, CHLD, CHLI 1, and CHLI 2 were

identified by screening the membrane-immobilized DNA of a Chlamydo-

monas BAC library (Lefebvre and Silflow, 1999) obtained from Clemson

University Genomics Institute (http://www.genome.clemson.edu/) using

the same probes as described for RNA detection. Hybridization of the

membranes was performed using the protocol provided by the Clemson

University Genomics Institute. Positive BAC clones that contained the

complete genes (17G11-CHLH 1, 26C5-CHLD, 35P13-CHLI 1, and 5J15-

CHLI 2) were obtained from Clemson University Genomics Institute. DNA

was isolated using the protocol of the BAC clone providers (http://

www.genome.clemson.edu/protocols.shtml).

Cloning and Sequencing of CHLD

Genomic sequence data (http://genome.jgi-pst.org/Chlre3/Chlre3.

home.html) provided information on restriction sites used for cloning of

CHLD. An 8.2-kb fragment containing the entire CHLD gene was obtained

by digestion of DNA of BAC clone 26C5 with NheI and HpaI. The resulting

8.2-kb fragment after blunt ending was ligated into the EcoRV site of the

pBluescript SKþ/� vector (Stratagene). The clone containing the correctinsert was verified by partial sequencing and restriction analysis. DNA of

this clone was tested for its ability to complement mutants defective in

chlorophyll synthesis upon transformation. For the identification of the

mutations in two mutants that could be complemented by the CHLD gene,

DNA was extracted from mutant strains chlD-1 and chlD-2 using the CTAB

method (von Gromoff et al., 1989). The sequence of the CHLD mutant

alleles was determined after amplification of segments that comprised the

entire CHLD coding region, using six primer pairs. The upstream (A) and

downstream (B) primers were as follows: 1A, 59-CACAACCAGGAC-

AGCCTA-39; 1B, 59-ATCGAAGCCCTTGGAGTC-39; 2A, 59-TGTCTGAG-

GAGGACTCCA-39; 2B, 59-GGGCTGGAACACAGTCTT-39; 3A, 59-AGG-

GCAAGACTGTGTTCCA-39; 3B, 59-CGATGGTCACGTCCTTCA-39; 4A,

59-TCATCCTGGCTCGCGAGTA-39; 4B, 59-CTTGTTCACACGCTCACG-39;

5A, 59-TGTCTCGAGCGAGTTGCTG-39; 5B, 59-ATGCGCTGTACTCACT-

GTCTG-39; 6A, 59-CTCATCTTCAGCGACGAC-39; 6B, 59-ACCTCAGTCA-

CTCGGCA-39.

The reactions were performed for 35 cycles of 1 min at 948C, 1 min at 50

or 558C, and 2.5 min at 728C with Pfu DNA polymerase (Fermentas), which

possesses proofreading activity. Resulting overlapping PCR products

were subcloned into the pGEM-T vector system (Promega) and se-

quenced. Sequencing was performed by the dideoxynucleotide chain

termination method (Sanger et al., 1977) using the ALF DNA analyzing

system (Amersham Biosciences Europe).

Nucleic Acid Manipulations

As the basic plasmid used for all promoter activity tests, we employed

pCB412, which is pARG7.8cos (Purton and Rochaix, 1995) with a

polylinker inserted into the unique NruI site. All constructs tested were

located on this vector and have been described previously (von Gromoff

et al., 2006).

Sources of Porphyrins

The MgProto used came either from Frontier Scientific or was prepared as

described previously (Kropat et al., 1997). This latter MgProto was kindly

provided by U. Oster. The concentration of MgProto was determined in

ether by spectrophotometric measurements using a millimolar extinction

coefficient of 308 at 419 nm (Falk, 1964). The concentration of hemin

(Sigma-Aldrich) was determined in a solvent consisting of 66.5% ethanol,

17% acetic acid, and 16.5% water (v/v/v) using a millimolar extinction

coefficient of 144 at 398 nm (Thomas and Weinstein, 1990). Other

metalated porphyrins were from Frontier Scientific. Their concentrations

were determined as specified by the supplier. For tests in vivo, tetrapyr-

roles were dissolved in dimethyl sulfoxide and used at the final concen-

tration indicated. The final dimethyl sulfoxide concentration in the culture

medium in all cases was 0.25%.

Pigment Analyses

Porphyrin steady state levels were determined in cells grown in liquid.

Cells were harvested and stored at�808C prior to rupture by sonication inan ice bath. Porphyrins were extracted in three steps with (1) methanol, (2)

K-phosphate buffer, and (3) a mixture of acetone: methanol: 0.1 N NH4OH

(10:9:1, v/v) (Alawady and Grimm, 2005). Aliquots of the supernatant were

separated by HPLC (model 1100; Agilent) on an RP 18 column (Novapak

C18, 4 mm particle size, 3.9 3 150 mm; Waters Chromatography) at a flow

rate of 1 mL/min in a methanol/0.1 M ammonium acetate, pH 5.2, gradient

and eluted with a linear gradient of solvent B (90% methanol and 0.1 M

ammonium acetate, pH 5.2) and solvent A (10% methanol and 0.1 M

ammonium acetate, pH 5.2). The eluted samples were monitored by a

564 The Plant Cell

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fluorescence detector at an excitation wavelength of 405 nm and emis-

sion wavelength of 620 nm for Proto and an excitation wavelength of 420

nm and emission wavelength of 595 nm for Mg-porphyrins. The porphy-

rins and metalloporphyrins were identified and quantified by comparison

with authentic standards purchased from Frontier Scientific. The content

of noncovalently bound heme was determined after removal of chloro-

phyll from Chlamydomonas cells by intensive washing with ice-cold

alkaline acetone containing 0.1 N NH4OH (9:1; v/v) (Weinstein and Beale,

1984). Heme was then extracted with acidic acetone containing 5% HCl,

transferred to diethyl ether, concentrated, and washed on a DEAE-

Sepharose column. Absorbance was monitored at 398 nm using a milli-

molar extinction coefficient of 144. The concentration of heme standard

solutions was determined spectrophotometrically (Weinstein and Beale,

1983).

Determination of CHL Activity

For determination of CHL activity, Chlamydomonas cells were grown in

500 mL of liquid TAP medium to a density of 3 to 5 3 106 cells per mL,

harvested by centrifugation, and resuspended in homogenization buffer

(0.5 M sorbitol, 0.1 M Tris-HCl, 1 mM DTT, and 0.1% BSA, pH 7.5). The

cells were disrupted by sonication in an ice bath. The cell extracts were

centrifuged at 15,000g for 30 min and the supernatants were used for

enzyme assays. CHL was assayed as described by Walker and Weinstein

(1991). The assay mixture contained 40 mM MgCl2 and 20 mM ATP. The

reaction, performed at 288C in individual opaque tubes, was started by

adding 100 mM Proto and stopped after 5, 10, 15, 30, and 60 min by

freezing the tubes in liquid nitrogen. Samples from each enzyme assay

were extracted and the products quantified according to methods de-

scribed above.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers AJ307055 (CHLH) and AF343974

(CHLI1). The other genes may be found as gene models in the Chlamy-

domonas genome sequence (http://genome.jgi-psf.org/Chlre3/Chlre3.

home.html) (CHLI2, gene model C_110312; CHLD, gene model C_60238).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Test of the Effect of Inhibitors of Cytosolic

and Organellar Protein Synthesis on the Light Activation of Mg-

Chelatase Genes.

Supplemental Figure 2. Assay of Tetrapyrrole Pool Levels in Mg-

Chelatase Mutants after a Shift from Dark to Light.

Supplemental Figure 3. Assay of Tetrapyrrole Pool Levels in Wild-

Type Cells That, before a Dark-to-Light Shift, Were Incubated with

Hemin in the Dark for 12 h.

ACKNOWLEDGMENTS

We thank Krishna Niyogi and Marilyn Kobayashi (University of California,

Berkeley, CA), in whose laboratory the mutants were isolated during a

sabbatical stay of C.F.B. in Berkeley. We also thank Ulrike Oster for

providing us with MgProto and Wolfhart Rüdiger (both University of

Munich) for helpful suggestions. Michael Schroda’s (University of Frei-

burg) valuable suggestions and his critical reading of the manuscript are

greatly appreciated. This work was supported by a grant from the

Deutsche Forschungsgemeinschaft to C.F.B.

Received July 31, 2007; revised February 15, 2008; accepted February

29, 2008; published March 25, 2008.

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Heme, a Plastid-Derived Regulator of Nuclear Gene ...b Institut fu¨r Biologie/Pﬂanzenphysiologie, Humboldt Universita¨t, D-10115 Berlin, Germany To gain insight into the chloroplast-to-nucleus