Botanical Studies (2006) 47: 389-395.

*

Corresponding author: E-mail: yycharng@gate.sinica.edu.

tw; Fax: 886-2-26515600.

INTRODUCTION

Heat-shock proteins (Hsps) are transiently induced

by elevated temperature to confer protection against

the detrimental effect of heat stress, a phenomenon

known as heat shock (HS) response (Lindquist, 1986).

Conserved Hsps, such as Hsp100, Hsp90, Hsp70, Hsp60,

and the small Hsps, have been well characterized as

molecular chaperones, responsible for the acquisition of

thermotolerance by maintaining the homeostasis of protein

folding in cells (Vierling, 1991; Parsell and Lindquist,

1993). With the progress of genomic and functional

genomic research, novel Hsps have been identified from

various species (Gasch et al., 2000; Helmann et al., 2001;

Shockley et al., 2003; Pysz et al., 2004; Rizhsky et al.,

2004; Busch et al., 2005). However, the functions of many

of these novel Hsps are unknown, thus hindering a further

understanding of this important physiological process.

In an attempt to discover the unique features in the

plant HS response, we have previously identified and

characterized a novel Hsp gene, Hsa32, which is highly

conserved in land plants but not present in most other

organisms (Liu et al., 2006). Suppression of Hsa32

expression by T-DNA insertion or RNAi transgene led to

significant defects in acquired thermotolerance during a

long recovery period after acclimation, which suggests an

important role for the protein in a protection mechanism

needed in plants (Charng et al., 2006). How Hsa32 exerts

its function is currently unknown. The gene encodes

an HS-induced 32-kD protein dissimilar to any well-

characterized Hsps but weakly similar (about 35%) to

the bacterial (2R)-phospho-3-sulfolactate (PSL) synthase

(Graham et al., 2002). The structural homology to PSL

synthase provides a possible clue.

In archaeal methanogens, PSL synthase or ComA cata-

lyzes the first step in the biosynthetic pathway of coen-

zyme M (CoM), which serves as a cofactor for methane

production (Graham et al., 2002). In this reaction, PSL is

formed by the stereospecific addition of sulfite to phos-

phoenolpyruvate. Since plants do not have the other CoM

biosynthesis genes, it was suggested that the plant PSL

synthase-related protein is involved in the biosynthesis of

sulfoquinovosyl diacylglycerol (SQDG) (Graham et al.,

BIOChemISTRy

hsa32, a phosphosulfolactate synthase-related heat-

shock protein, is not involved in sulfolipid biosynthesis

in Arabidopsis

Nai-Yu LIU, Wan-Jen HSIEH, Hsiang-Chin LIU, and Yee-Yung CHARNG*

Agricultural Biotechnology Research Center, Academia Sinica, Taipei, TAIWAN 11529, Republic of China

(Received November 1, 2005; Accepted March 15, 2006)

ABSTRACT

. Hsa32 is a novel heat-shock protein (Hsp) mainly found in land plants. Recently, it was

shown to be essential for acquired thermotolerance following a long recovery after acclimation heat-shock

(HS) treatment. Without Hsa32, Arabidopsis mutant plants become more sensitive to severe HS than wild-

type plants due to faster decay of a previously acquired protection. Sequence homology showed Hsa32 to be

a phosphosulfolactate synthase-related protein, and it was proposed to be involved in the biosynthesis of sul-

foquinovosyl diacylglycerol (SQDG), one of the major sulfur-containing glycolipids in the chloroplast thyla-

koid membrane. Currently, Sqd1 and Sqd2 are known to catalyze the consecutive reactions in the biosynthetic

pathway of the sulfolipid. In this study, we examine Hsa32¡¦s possible involvement in an alternative pathway

that bypasses Sqd1. Our analysis of the wild type and Hsa32 T-DNA knockout mutant plants revealed no

significant differences in SQDG accumulation. In addition, the Arabidopsis mutant with a disrupted Sqd1

gene did not synthesize SQDG, which discounts the existence of an alternative pathway. The Sqd1 and Sqd2

knockout mutants, both lacking SQDG, did not show the same defect in acquired thermotolerance as did the

Hsa32 null mutant, which suggests that the sulfolipid level is not related to the HS-sensitive phenotype. Our

data suggest that Hsa32 is not involved in SQDG biosynthesis.

Keywords: Arabidopsis; Sulfolipid; Thermotolerance.

Abbreviations: HS, Heat-shock; UDP-SQ, uridine diphosphate-sulfoquinovose; SQDG, sulfoquinovosyl dia-

cylglycerol.

390

Botanical Studies, Vol. 47, 2006

2002). SQDG is a sulfur-containing glycolipid specifi-

cally associated with the photosynthetic membranes of

plants and most photosynthetic bacteria (Benning, 1998).

The major function of SQDG is the substitution of anionic

phospholipids under phosphate-limited growth conditions

( Yu et al., 2002).

Recently, Benning (1998) demonstrated a 2-step bio-

synthetic reaction sequence of SQDG, the sugar-nucleo-

tide pathway (Figure 1): uridine diphosphate-SQ (UDP-

SQ) is formed from UDP-Glc and sulfite and catalyzed

by Sqd1, the UDP-SQ synthase (Essigmann et al., 1998;

Sanda et al., 2001), then SQ is transferred from UDP-SQ

onto DG, and catalyzed by Sqd2, the SQDG synthase (Yu

et al., 2002). Insertion of an Agrobacterium T-DNA into

Sqd2 in Arabidopsis inhibits sulfolipid formation in the

knockout mutant (Yu et al., 2002). Although a deletion

mutant of Sqd1 of the unicellular alga Chlamydomonas

reinhardtii was shown to be devoid of SQDG (Riekhof

et al., 2003), the existence of an alternative pathway for

UDP-SQ synthesis in higher plants cannot be excluded.

Actually, about 40 years ago, Davies et al. (1966) pro-

posed that phosphosulfolactate was one of the intermedi-

ates of SQDG biosynthesis in the so-called sulfoglycolytic

pathway (Figure 1). Recent reports revealed that SQDG

accumulation increases in leaves of drought-resistant

plants under high temperature (Taran et al., 2000) and

that SQDG is involved in the structural integrity and heat

tolerance of photosystem II (Sato et al., 2003), which sug-

gests that synthesis of SQDG might be increased to confer

protection in plants under heat stress. Therefore, it is of

interest to know whether the plant PSL synthase-related

protein (i.e. Hsa32) participates in an HS-inducible SQDG

biosynthetic pathway.

In this study, we examined the role of Hsa32 by genetic

and biochemical analysis of the Arabidopsis Sqd1 and

Hsa32 T-DNA insertion mutants. The Arabidopsis Sqd1

knockout mutant did not accumulate a measurable amount

of SQDG under normal and heat stress conditions. The

sulfolipid level in wild-type and hsa32 knockout mutant

plants was also not affected by HS treatment, which

suggests no alternative pathway for UDP-SQ synthesis

under normal or heat stress conditions and that Hsa32 is

not involved in the biosynthesis of sulfolipids in higher

plants. Moreover, the Arabidopsis plants without SQDG

accumulation did not exhibit an HS-sensitive phenotype

as did the Hsa32 knockout plants, which rules out the

hypothesized role of Hsa32 as a sulfolipid biosynthesis-

related enzyme.

mATeRIALS AND meThODS

Sequence alignment

The amino acid sequences of Arabidopsis Hsa32

(NP_567623) and the PSL synthase of Methanococcus

jannaschii (Q57703) were aligned by use of the AlignX

(InforMax) program with the default setting (Figure 2).

Plant materials and growth conditions

The Arabidopsis T-DNA insertion lines of Sqd1,

Sqd1-KO (SALK_016799), and Sqd2, Sqd2-KO

(SALK_105492), generated in a Col-0 background

Figure 1. Schematic pathways of SQDG biosynthesis. Two distinct pathways have been proposed for the synthesis of UDP-SQ, the

precursor of the sulfolipid head group. The sulfoglycolytic pathway (A) was drawn according to Davis et al. (1966) with modification.

The enzymes for this pathway have not been identified and the step possibly catalyzed by Hsa32 is shown. The sugar-nucleotide path-

way (B) was drawn according to Benning (1998).

LIU et al. ¡X Hsa32 is not involved in sulfolipid biosynthesis

391

(Alonso et al., 2003), were obtained from the Arabidopsis

Biological Resource Center (Columbus, OH). The

Hsa32 T-DNA insertion mutant hsa32-1 was obtained as

previously described (Charng et al., 2006). The

location

of the T-DNA insertion was confirmed by PCR and

DNA sequencing. Seeds

of the mutants and wild type

Arabidopsis thaliana (Col-0) were sown on 0.8% (w/v)

agar-solidified MS medium supplemented with 1% (w/v)

sucrose and kept at 4¢XC for at least 3 days. Plants were

grown in a growth chamber with cool white fluorescent

light of 130 £gmol m

-2

¡Ps

-1

under 14-h light/10-h

dark and

23¢XC/18¢XC cycles. For the acquired thermotolerance test,

Arabidopsis seedlings were grown at 24

o

C with 16-h

light (130 £gmol m

-2

s

-1

) in a Petri dish (90 ¡Ñ 15 mm) with

25 mL of solid medium (25 mL of 0.5 ¡Ñ MS containing

0.8% agar and 1% sucrose). The plate containing 3-d old

seedlings was submerged in a water bath for HS treatment

as described in the figure legends. During recovery from

each HS treatment, the plate was removed from the water

bath and kept at the previous growth conditions under the

same light/dark cycles until photographed.

RNA isolation and analysis

Total RNA was isolated from frozen samples with

use of TRIZOL reagent (Invitrogen). The expression

of Hsa32 was determined by northern blot analysis as

previously described (Li et al., 2003). The expression of

Sqd1 was detected by RT-PCR with the forward primer 5¡¦-

GCGCATCTACTTTCAGCTTCATGCCCTTC-3¡¦ and

reverse primer 5¡¦-TTATGTGGTCATGGACTTAGTCT

TGACGC-3¡¦, and that of Sqd2 with the forward primer

5¡¦-GCTTCATCTCCCGGAGTTATG-3¡¦ and reverse

primer 5¡¦-TCCAATGAAAGCAATCCGAGC-3¡¦. RT-

PCR experiments were performed as described previously

(Wang et al., 2001). All RT-PCR products were confirmed

by direct sequencing.

Immunoblot analysis

The total protein of plant samples was extracted by

use of Tris-HCl buffer (60 mM, pH 8.5, containing

2% SDS, 2.5% glycerol, 0.13 mM EDTA, and 1%

protease inhibitor cocktail). The protein amount was

measured by use of DC Protein Assay reagents (Bio-

Rad), with bovine serum albumin used as a standard. For

immunoblot analysis, protein was separated on SDS-

polyacrylamide gel electrophoresis (SDS-PAGE) with a

precast minigel assembly (NuPAGE 4-12% Bis-Tris gel

+ MOPS SDS running buffer; Invitrogen) and transferred

onto a nitrocellulose membrane for antibody probing.

The polyclonal antibody against Arabidopsis Hsa32 was

prepared by immunizing rabbits with purified recombinant

protein (Charng et al., 2006). The polyclonal antibody

against rice class-I sHsp was kindly provided by Dr. Chu-

Yung Lin (National Taiwan Univ.). The amount of antigen

was detected by use of the Super Signal West Dura

Extended Duration Substrate system (Pierce). Following

chemiluminescence detection, the membrane was stained

with 0.1% (w/v) amido black to ensure equal loading of

protein.

Lipid analysis

The

35

S-labeled SQDG in the leaves of the Arabidopsis

wild-type and mutant plants were analyzed by thin layer

chromatography (TLC) as previously described (Yu et al.,

2002).

ReSULTS AND DISCUSSION

hsa32 is similar to PSL synthase in primary

structure

To elucidate the molecular function of Hsa32, we

started from a structure comparison approach. Hsa32 is not

Figure 2. Alignment of the deduced amino acid sequences of Arabidopsis Hsa32 (AtHsa32) and M. jannaschii PSL synthase

(MjPSLS). Residues that are identical between the two sequences are shown in white letters with a black background; chemically

conserved residues are shown with a gray background. Gaps (indicated by hyphens) have been introduced to give a better alignment.

Based on the three dimensional structure and sequence of the M. jannaschii enzyme (Wise et al., 2003), the catalytic lysine residue

(K137) is indicated by an arrow. The proposed binding residues for PEP (W46 and T76), sulfite (R170 and R244¡¦), and magnesium

(E103, E133, E168, and E205) are indicated by ^, +, and

*

, respectively, underneath the alignment.

392

Botanical Studies, Vol. 47, 2006

homologous to any previously identified Hsps. The plant

protein is most similar to a PSL synthase as previously

reported (Graham et al., 2002). Alignment of PSL

synthase and Hsa32 revealed conserved residues spanning

the entire protein, including the lysine residue of the

catalytic site (Figure 2). However, the degree of homology

is low. The two proteins share only 18% identity and

38% similarity. Nevertheless, the sequence homology has

led to a postulation that Hsa32 contributes to sulfolipid

biosynthesis (Graham et al., 2002) in the sulfoglycolytic

pathway (Figure 1).

Many amino acid residues in the substrate binding sites

of PSL synthase from M. jannaschii (Wise et al., 2003) are

not conserved in the Hsa32 sequence. Of the eight amino

acid residues proposed to interact with the substrates phos-

phoenolpyruvate, sulfite, and magnesium in PSL synthase,

only two, E103 and E205, which bind to magnesium ions,

are conserved in the plant sequence (Figure 2). The W46

and T76 residues, proposed to bind phosphoenolpyruvate,

are also not conserved in Hsa32. Thus, Hsa32 might not

necessarily have the same catalytic activity as the M. jan-

naschii enzyme. No direct evidence to date suggests that

Hsa32 can synthesize PSL in vitro or in vivo.

Synthesis of SQDG is not affected in the hsa32

knockout mutant

To test whether Hsa32 is involved in the biosynthesis

of sulfolipids (Figure 1), a genetic approach was used to

directly examine SQDG in a knockout mutant of Hsa32,

hsa32-1, caused by a T-DNA insertion at the third exon

(Figure 3A). The hsa32-1 mutant did not respond to HS

treatment at either the transcriptional (Figure 3B) or trans-

lational (Figure 3C) levels as did the wild type, which

indicates that the T-DNA insertion results in a null mutant

by disrupting the integrity of the gene.

Since Hsa32 is a single-copy gene in the Arabidopsis

genome, we thought the SQDG level would be affected if

Hsa32 contributed significantly to the biosynthesis of the

lipid under heat stress. Therefore, we examined the SQDG

synthesis in the mutant under normal conditions and after

HS treatment by TLC analysis of the lipids extracted from

detached leaves fed with

35

S isotope-labeled sulfate. In the

wild-type leaf,

35

S-labeled sulfate was converted into

35

S-

labeled SQDG (Figure 4) as was previously reported (Yu

et al., 2002). However, the level of SQDG was not signifi-

cantly affected by HS treatment in the wild type. The level

of SQDG was likely maintained at a steady level under

heat stress by up-regulating the sulfoglycolytic pathway

with the suppression of another pathway. If so, then the

Figure 3. Disruption of Arabidopsis Hsa32 by T-DNA insertion

led to a null mutation. The location of the T-DNA insertion in

the hsa32-1 (GABI-Kat 268A08) mutant is indicated schemati-

cally (A). Black bars indicate exons. The transcription and trans-

lation of Hsa32 in response to heat stress (37

o

C, 2 h) in hsa32-1

and wild-type (Wt) were revealed by northern (B) and western

blotting (C). The level of clas s I small Hsp (s Hsp-CI) was

shown as a HS positive control. Rubisco large subunit stained

by amido black was shown to ensure equal loading.

Figure 4. TLC analysis of sulfolipid in hsa32-1 leaves. Lipids

were extracted from leaves treated with [

35

S]SO

4

2-

of the wild-

type (Wt) and hsa32-1 plants without (kept at room temperature,

RT) or with HS treatment (37

o

C for 2 h), then separated on a

TLC plate, and [

35

S]-labeled SQDG was visualized by autoradi-

ography. The SQDG bands are indicated by arrows.

LIU et al. ¡X Hsa32 is not involved in sulfolipid biosynthesis

393

level of SQDG in hsa32-1 should decline. However, the

sulfolipid level did not significantly change before, dur-

ing, or after HS treatment, and no significant difference

between the wild-type and the mutant plants was observed

either (Figure 4).

Knockout mutant of Sqd1 does not synthesize

SQDG under normal and hS conditions

To further confirm our results, we analyzed the

Arabidopsis Sqd1 knockout mutant to determine the

existence of an alternative pathway of SQDG biosynthesis.

Because Sqd1 is a single-copy gene in the Arabidopsis

genome, the Sqd1 knockout mutant plant should not

synthesize SQDG if the sugar-nucleotide pathway (Figure

1) is the sole pathway. To test this hypothesis, we obtained

and confirmed a T-DNA knockout line with a disrupted

Sqd1 gene (Figure 5A). A T-DNA knockout mutant of

Sqd2 essential for SQDG biosynthesis (Yu et al., 2002)

was employed here as a control. RT-PCR analysis showed

that the T-DNA insertion lines Sqd1-KO and Sqd2-KO

did not accumulate corresponding transcripts (Figure

5B), which confirmed the disruption of the genes. Neither

mutant showed any significant phenotypic difference as

compared with the wild type (data not shown), a result

consistent with a previous report that the absence of

SQDG did not result in any defect in development or

growth under normal conditions (Yu et al., 2002). Then,

we analysed the level of SQDG in each of these mutants

Figure 5. Disruption of Arabidopsis Sqd1 and Sqd2 by T-DNA

insertion. The locations of T-DNA insertion in the Sqd1-KO

(SALK_016799) and Sqd2-KO (SALK_105492) mutants are

indicated s chematically (A). Black bars indicate exons. The

transcript levels of Sqd1 (lane 1-3) and Sqd2 (lane 4-6) in the

wild-type (lane 1 and 4), Sqd1-KO (lane 2 and 5), and Sqd2-KO

(lane 3 and 6) plants were revealed by RT-PCR (B). The bands

representing RT-PCR products of Sqd1 (1,431 bp) and Sqd2 (444

bp) are indicated.

Figure 6. TLC analysis of sulfolipid in Sqd1 knockout mutant. Lipids were extracted from leaves fed with [

35

S]SO

4

2-

of the wild-type

(Wt), Sqd1-KO (sqd1), and Sqd2-KO (sqd2) mutants without (RT) or with HS (37

o

C for 2 h) treatment, then separated on a TLC plate

and visualized by use of £\-naphthol, which primarily stains glycolipids (A), or by autoradiography to show [

35

S]-labeled SQDG (B).

394

Botanical Studies, Vol. 47, 2006

by TLC. Under normal and HS conditions SQDG was

accumulated in the wild-type Arabidopsis but not in the

Sqd1-KO or Sqd2-KO mutant leaves (Figure 6). Thus,

evidence of an alternative pathway is lacking, and Sqd1 is

apparently involved in the major if not the sole pathway

of SQDG biosynthesis. A complementation experiment is

needed to ensure that the absence of SQDG in the Sqd1-

KO mutant was not caused by a secondary mutation.

However, this is beyond the scope of our study and hence

was not done.

Relation between SQDG level and hS sensitivity

To further confirm that the thermotolerance defect of

hsa32-1 is not due to any minor change in sulfolipid level,

we compared the thermotolerance of the Sqd1-KO, Sqd2-

KO, and hsa32-1 mutants. As reported earlier, hsa32-1

plants became less tolerant than the wild type to severe HS

challenge after a long recovery, despite containing normal

levels of SQDG; however, the Sqd1-KO and Sqd2-KO

plants, which did not accumulate measurable SQDG, still

remained tolerant to the HS treatment (Figure 7). Thus,

the sulfolipid level was not associated with the defective

phenotype in the acquired thermotolerance of hsa32-1.

Taken together, our data indicate that Arabidopsis

Hsa32 is not involved in the biosynthesis of SQDG.

Acknowledgements. We thank Yu Kuei for technical sup-

port. We also thank Dr. Christopher Benning for providing

guidance in analysis of SQDG. Drs. Tzyy-Jen Chiou and

Kuo-Chen Yeh are appreciated for their discussion of our

results and reading of the manuscript. This study was sup-

ported by the National Science Council (91-3112-P-001-

036-Y and 94-2311-B-001-058), Taiwan, ROC.

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