A role for SENSITIVE to FREEZING2 in protecting chloroplasts against freeze-induced damage in Arabidopsis

Article (PDF Available)inThe Plant Journal 55(5):734-45 · June 2008with24 Reads
DOI: 10.1111/j.1365-313X.2008.03549.x · Source: PubMed
The sensitive to freezing2 (SFR2) gene has an important role in freezing tolerance in Arabidopsis thaliana. We show that homologous genes are present, and expressed, in a wide range of terrestrial plants, including species not able to tolerate freezing. Expression constructs derived from the cDNAs of a number of different plant species, including examples not tolerant to freezing, are able to complement the freezing sensitivity of the Arabidopsis sfr2 mutant. In Arabidopsis the SFR2 protein is localized to the chloroplast outer envelope membrane, as revealed by the analysis of transgenic plants expressing SFR2 fusions to GFP, by confocal microscopy, and by the immunological analysis of isolated chloroplasts treated with thermolysin protease. Moreover, the chloroplasts of the sfr2 mutant show clear evidence of rapid damage after a freezing episode, suggesting a role for SFR2 in the protection of the chloroplast.
A role for SENSITIVE TO FREEZING2 in protecting
chloroplasts against freeze-induced damage in Arabidopsis
Nicolas Fourrier
, Jocelyn Be
, Enrique Lopez-Juez
, Adrian Barbrook
, John Bowyer
, Paul Jarvis
Gareth Warren
and Glenn Thorlby
School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK,
Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, UK, and
Department of Biochemistry, University of Cambridge, Hopkins Building, Downing Site, Cambridge CB2 1QW, UK
Received 4 March 2008; accepted 17 April 2008; published online 11 July 2008.
For correspondence (fax +44 178 443 4326; e-mail g.thorlby@rhul.ac.uk).
Unfortunately Dr Warren and Prof. Bowyer died before the completion of this work.
The SENSITIVE TO FREEZING2 (SFR2) gene has an important role in freezing tolerance in Arabidopsis thaliana.
We show that homologous genes are present, and expressed, in a wide range of terrestrial plants, including
species not able to tolerate freezing. Expression constructs derived from the cDNAs of a number of different
plant species, including examples not tolerant to freezing, are able to complement the freezing sensitivity of
the Arabidopsis sfr2 mutant. In Arabidopsis the SFR2 protein is localized to the chloroplast outer envelope
membrane, as revealed by the analysis of transgenic plants expressing SFR2 fusions to GFP, by confocal
microscopy, and by the immunological analysis of isolated chloroplasts treated with thermolysin protease.
Moreover, the chloroplasts of the sfr2 mutant show clear evidence of rapid damage after a freezing episode,
suggesting a role for SFR2 in the protection of the chloroplast.
Keywords: Freezing tolerance, SFR2, Arabidopsis, chloroplast.
Freezing tolerance is a widely-distributed trait in the plant
kingdom, but it is generally absent from species native to
tropical and subtropical regions. Even in crop species with
qualitative tolerance to freezing, there is frequently a quan-
titative deficiency that has an impact on their agronomy.
There are therefore applied, as well as fundamental, reasons
to desire an understanding of freezing tolerance.
Complementing a long history of physiological and bio-
chemical approaches, much work over the last two decades
has focussed on gaining a molecular genetic understanding
of freezing tolerance. By investigating changes in gene
expression during cold acclimation (reviewed by Thom-
ashow, 1999), important parts of the signal transduction
pathway have been elucidated, culminating in the quantita-
tive manipulation of freezing tolerance with transcription
factors (Chinnusamy et al., 2003; Gilmour et al., 2000; Jaglo-
Ottosen et al., 1998; Kasuga et al., 1999). In parallel with this
approach, components of the cold-signalling pathway have
been identified, using an elegant mutant screening strategy
with plant lines containing stress-responsive molecular
reporters (Ishitani et al., 1997). This has revealed a number of
novel components of the cold signal transduction pathway
(Xiong et al., 2002; Zhu et al., 2005).
Mutations have also been identified on the basis of their
direct effect on the freezing-tolerance phenotype. The esk
mutants display elevated levels of freezing tolerance in the
absence of cold acclimation (Xin and Browse, 1998); their
molecular characterization may identify genes that make
self-sufficient individual contributions to freezing tolerance.
Mutants that are deficient in freezing tolerance have also
been isolated. The frs1 mutation was shown to be an allele of
ABA3 (Llorente et al., 2000), confirming the need for ABA
signalling during cold acclimation. The sensitive to freezing
(sfr) mutants (Warren et al., 1996) display a range of lesions
following freezing, and identify a set of eight genes that are
important for freezing tolerance, and that do not correspond
to genes previously known or suspected to be involved in
this phenomenon (Thorlby et al., 1999).
The SFR2 gene was recently identified as a constitu-
tively expressed b-glucosidase that is important for
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The Plant Journal (2008) 55, 734–745 doi: 10.1111/j.1365-313X.2008.03549.x
freezing tolerance in Arabidopsis (Thorlby et al., 2004).
The protein belongs to the Family-1 b-glycosidases
and contains the highly conserved peptide active site
motifs, TFNEP and I/VTENG, and additionally the
GYIFWTISDNWEW (a variant of the nearly invariant
GYFAWSLXDNFEW) motif (Xu et al., 2004). The two
glutamate residues within the active site motifs serve as
the acid base catalyst and the nucleophile of the cleavage
reaction (Davies and Henrissat, 1995).
In Arabidopsis, b-glycosidases comprise a large multi-
gene family of 48 members. Forty-seven members share a
common evolutionary origin, whereas SFR2 belongs to a
distinct lineage, and with the exception of the three
conserved motifs, it shares little sequence similarity with
other family members (Xu et al., 2004). Indeed, SFR2 is
more closely related to bacterial glucosidases than to other
members of the Arabidopsis multigene family (Thorlby
et al., 2004). Family-1 b-glycosidases are involved in a wide
range of biological reactions, where they affect various
characteristics of glycosylated moieties, including their
reactivity, solubility and transport (Li et al., 2001). A wide
range of roles have been postulated, including defence
against pathogens (Cicek and Esen, 1998), cell wall biosyn-
thesis (Li et al., 2001), antioxidant release (Chong et al.,
2002) and the stress-induced release of phytohormones (Lee
et al., 2006). However, not all of the substrates cleaved by
Family-1 b-glycosidases are carbohydrates (Coutinho et al.,
2003; Raychaudhuri and Tipton, 2003). The multitude of
possible mechanisms and potential substrates for the SFR2
enzyme make predictions about its role in protecting plants
from damage during freezing difficult, and the mechanism
of this is yet to be determined.
An intriguing question is whether SFR2 function is
deficient in species that qualitatively lack freezing tolerance.
Initial observations suggested that expressed sequence tag
(EST) sequences with the potential to encode proteins with
high homology to the SFR2 protein were present in plants
over a wide phylogenic distribution, including species that
were sensitive as well as tolerant to freezing. We decided to
characterize representative members of this family of
orthologous genes, and to test their ability to functionally
complement the Arabidopsis sfr2 mutation and restore
freezing tolerance. To shed light on the role of SFR2 in
freezing tolerance, we also investigated the subcellular
localization of the protein, and the mode of damage in the
Arabidopsis mutant.
SFR2-like proteins are widely distributed throughout land
Initial analysis of EST sequences in the NCBI database
indicated that numerous species potentially produce SFR2-
like proteins. These EST clones were from a wide range of
plant species, including both angiosperms, representing
dicotyledonous and monocotyledonous species, as well as
gymnosperms. We chose a representative set from which to
isolate full-length cDNA clones. Examples were selected
from a gymnosperm (Pinus taeda) as well as from both
dicotyledonous (Glycine max and Solanum esculentum) and
moncotyledonous (Oriza sativa and Zea mays) species.
Included in this set are species regarded as freezing sensitive
(Z. mays, O. sativa and S. esculentum), as well freezing-
tolerant examples. In the case of O. sativa, we were able to
confirm the gene sequence using information from the rice
genome project.
Analysis of the proteins encoded by the isolated cDNAs
showed a high degree of similarity between all of the
selected proteins (Fig. 1). Homology was strongest in the
central region of the protein. The active-site peptide
motifs (TFNEP and I/VTENG) are fully conserved in
AtSFR2, LeSFR2, GmSFR2 and PtSFR2, and only differ in
OsSFR2 and ZmSFR2 by the substitution of Ile or Val,
respectively, for Thr in the TFNEP motif. The N- and
C-termini of the proteins are more divergent; however, the
N-terminus carries a stretch of 11 amino acids (FFFGLA-
TAPAH), at position 61 in AtSFR2, which is perfectly
conserved in all SFR2-like sequences. The similarity of the
SFR2-like proteins to the Arabidopsis SFR2 protein, and
the conservation of the active site motifs, suggest that the
catalytic function is likely to be conserved.
In order to assess the phylogenetic relationships of
SFR2-like proteins, we carried out searches of the
sequenced genomes of simple plant species. Analysis of
the genome sequences of the eukaryotic alga Chlamydo-
monas reinhardtii and the oxygenic prokaryote Gloeo-
bacter violaceus identified genes with the potential to
encode proteins with only weak similarity to SFR2,
whereas the genomes of the red algae Cyanidioschyzon
merolae and the marine diatom Thalassiosira pseudonana
did not contain genes with the potential to produce SFR2-
like proteins. The genes identified in C. reinhardtii and
G. violaceus were more similar to other members of the
Arabidopsis b-glycosidase gene family than they were to
the SFR2 gene, indicating that they were unlikely to
represent SFR2-like genes. Previous phylogenetic analysis
has demonstrated that the SFR2 protein is uniquely
divergent from other members of the 48-gene b-glycosi-
dase gene family in Arabidopsis (Thorlby et al., 2004). A
recent characterization of b-glycosidases in rice has also
demonstrated that the relevant SFR2-like gene has low
sequence similarity to other b-glycosidases in this species,
and is more similar to the Arabidopsis SFR2 gene
(Opassiri et al. , 2006). Phylogenetic analysis of putative
SFR2-like proteins (Fig. 2) shows that they form a distinct
clade, and that they are different from both the other
Arabidopsis b-glycosidases and the most similar proteins
SFR2 and freezing tolerance in Arabidopsis 735
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Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 734–745
encoded by a number of sequenced simple plant
genomes. The lack of a homologous sequence within
the cyanobacteria and simple plant species suggests that
the SFR2 gene is not likely to be derived from the
endosymbiotic bacterial genome associated with the
development of the chloroplast.
SFR2-like sequences from both the freezing-tolerant and
the freezing-sensitive species are able to complement
the sfr2 mutation in Arabidopsis
The presence of genes encoding proteins with high simi-
larity to the Arabidopsis SFR2 in species that are not freezing
Figure 1. Multiple protein sequence alignment of Solanum esculentum SFR2 (SeSFR2), Glycine max SFR2 (GmSFR2), Oriza sativa SFR2 (OsSFR2), Pinus taeda SFR2
(PtSFR2), Zea mays SFR2 (ZmSFR2) and Arabidopsis thaliana SFR2 (AtSFR2).
The sites of the active site peptide motifs (TFNEP and I/VTENG) are indicated (*). Fully conserved residues are boxed in black, and positively scoring groups in the
Gonnet Pam250 matrix are boxed in grey.
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tolerant, suggests a role for the protein that is separate from
its putative function in freeze protection. An obvious ques-
tion is whether SFR2-like genes from freezing-sensitive
species are able to complement the Arabidopsis sfr2
mutation. In order to test this, cDNA clones encoding the
SFR2-like proteins from G. max, O. sativa and P. taeda
were cloned into a plant expression vector, where their
expression was mediated by the cauliflower mosaic virus
35S promoter. These constructs were used to transform the
sfr2-i2 T-DNA-tagged allele, which has previously been
shown not to produce SFR2 protein (Thorlby et al., 2004).
Surprisingly, the SFR2-like sequences from both freezing-
tolerant (P. taeda) and freezing-sensitive (G. max and
O. sativa) species were able to functionally complement
the freezing-sensitivity lesion in Arabidopsis (Fig. S1). No
differences, either qualitative or quantitative, were observed
with regard to the ability of constructs from different species
to complement the sfr2 phenotype.
An interesting aspect of the sfr2 phenotype is that leaf
order dependent freezing sensitivity, which is found in
wild-type Arabidopsis (Takagi et al. , 2003), is preserved. As
with wild-type plants, younger leaves and meristematic
regions are more able to resist freezing than mature leaves
in the sfr2 mutant. In sfr2 at temperatures below )7C, the
whole plant including the meristematic region is killed,
whereas at more moderate freezing temperatures (above
)6.5C), the younger leaves and meristematic regions
usually survive the freezing regime, even though mature
leaves are killed, and the plant is able to recover (Fig. S2).
Nevertheless, the temperature at which damage is caused
to both young and mature tissues of sfr2 is higher (i.e. is
less cold) when compared with wild-type plants. At freez-
ing temperatures below )7.0C sfr2 plants are killed,
whereas only the mature leaves of wild-type plants are
damaged (Fig. S2).
As proof that the b-glycosidase activity of the AtSFR2
protein is required for freeze protection, an active site knock-
out was engineered by site-directed mutagenesis. The
Glu267 residue in AtSFR2, which acts as the acid/base
catalyst in the substrate hydrolysis reaction, and belongs to
the highly conserved peptide motif TFNEP of the active site
of Family-1 b-glycosidases, was altered to a Gly267. Glycine,
a small uncharged amino acid, located in the active site
pocket, is unlikely to modify the tertiary or quaternary
structure of AtSFR2. Wang et al. (1995) previously demon-
strated the applicability of this method when precisely
defining the function of the Glu residue in the TFNEP active
site of Agrobacterium faecalis b-glucosidase. The full-length
construct, with expression driven by the
35S promoter, was transformed into sfr2i2 mutant, and its
expression was verified using anti-SFR2 antibody. The
construct was not able to complement the freezing sensitiv-
ity, whereas a construct identical except for the G267E
mutation was able to complement freezing sensitivity
(Fig. 3). This suggests that b-glycosidase activity is essential
for the freezing tolerance role of the Arabidopsis protein,
and presumably also for the other SFR2-like proteins able to
complement the freezing lesion.
The SFR2 protein is localized to the chloroplast outer
In order to investigate a possible role for SFR2, the intra-
cellular localization of the protein was investigated using
GFP constructs. In silico analysis suggested the presence of
an N-terminal signal peptide, targeting the protein to the
secretory pathway (Thorlby et al., 2004), whereas proteo-
mics-based investigations have identified the SFR2 protein
in the chloroplast outer envelope fraction of both Arabid-
opsis (Dunkley et al., 2006; Ferro et al., 2003; Froehlich
et al., 2003) and Pisum sativum (Schleiff et al., 2003). Using
Gateway technology, a clone comprising a full-length copy
of the SFR2 gene (minus the stop codon) fused to EGFP
was constructed with the clone under the transcriptional
regulation of the 35S promoter. This clone was used to
Figure 2. Phylogenetic analysis of SFR2 and related protein sequences.
The neighbour-joining tree was constructed from a distance matrix of total-
character differences. Percentage bootstrap values over 60% are shown at
relevant nodes (1000 bootstrap replicates). Sequences used in the construction
of the tree are the SFR2-like genes of Solanum esculentum SFR2 (SeSFR2),
Glycine max SFR2 (GmSFR2), Oriza sativa SFR2 (OsSFR2), Pinus taeda SFR2
(PtSFR2), Zea mays SFR2 (ZmSFR2) and Arabidopsis thaliana SFR2 (AtSFR2).
The most similar procaryotic b-glycosidases are included: Thermosphaera
aggregans (AAD43138) and Thermosphaera sp. (CAA94187). Also included are
the most similar proteins from Gloeobacter violaceus (glr3230:3435813-
3437357 direct) and Chlamydomonas reinhardtii (C_9000168 in ChlamyDB;
http://www.chlamy.org). The Arabidopsis genes At4g21760 and At3g6012
were selected as being the Arabidopsis genes most similar to the G. violaceus
and C. reinhardtii sequences, respectively.
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generate stable transformants in wild-type Arabidopsis and
the two available SFR2 mutants, the original mutant car-
rying a point-mutation (sfr2-1) and the T-DNA-tagged allele
(sfr2-i2; Thorlby et al., 2004). Both mutant alleles of SFR2
were complemented by this construct, and their freezing
tolerance was restored (data not shown). The intracellular
localization of the tagged protein was investigated using
confocal microscopy. Control constructs showed the sol-
uble EGFP to have a cytoplasmic location (Fig. 4a,b). By
contrast, the tagged SFR2 protein, confirming the proteo-
mics-based studies, showed a pattern associated with
localization to the chloroplast envelope (Fig. 4c,d). No dif-
ferences in the localization of SFR2 were observed when
wild-type or mutant plants were transformed, or when
warm- or cold-grown plants were examined. Fluorescence
was most easily detected in the chloroplasts within sto-
matal guard cells. Interestingly, an unusual phenotype was
associated with transformation using the SFR2:GFP con-
struct: the chloroplasts appear bunched together (compare
panels b and c in Figure 4). This aggregation of chloro-
plasts seems to be a product of the GFP construct, as
expression of the SFR2 protein alone directed by the same
promoter did not lead to such a chloroplast phenotype
(data not shown). It is possible that the addition of GFP
causes the C-terminal region to face into the cytosol
where GFP–GFP interactions cause aggregation of the
To further investigate the mechanism of targeting of the
SFR2 protein to the chloroplast, we investigated whether the
N-terminal region is sufficient to target a passenger protein
to the organelle. A new construct in which only the first
31 N-terminal amino acids of the SFR2 protein were fused to
GFP showed the same chloroplast envelope localization
pattern (Fig. 4e,f). Fluorescence was considerably stronger
than with the full-length SFR2 construct, and aggregation of
chloroplasts was less pronounced. The localization was
again specific to the chloroplast envelope, suggesting that
the N-terminal region of SFR2 is sufficient to direct the
protein to this location.
In order to confirm the localization of SFR2, and to
establish its position in the chloroplast envelope system,
we used immunoblot analysis with purified chloroplasts
and an anti-SFR2 antibody directed against the C terminus
of the SFR2 protein (the antibody was raised against
residues 514–622/end of AtSFR2) (Thorlby et al. , 2004).
This confirmed the localization of SFR2 to the purified
chloroplast fraction (Fig. 5). Treatment of the isolated
chloroplasts with thermolysin protease before analysis
showed that SFR2 is partially sensitive to the protease, and
produced a band of reduced size. This implies that the
SFR2 protein is localized in the chloroplast outer envelope
membrane (where it is partially accessible to the protease),
and that its C-terminus, which is recognized by the
antibody, is protected from digestion by its integration
into or across the membrane, where thermolysin cannot
penetrate (Cline et al., 1984). Thermolysin digestion of
in vitro-translated SFR2 protein shows that, in the absence
of such native membrane association, SFR2 is fully
susceptible to thermolysin, and that the protected
fragment of the native protein is not the result of a
protease-resistant domain (Fig. 5). The similar molecular
weight of the in vitro synthesized protein to the endoge-
nous SFR2 protein in chloroplasts suggests that there is no
significant post-translational modification of the protein,
and that there is unlikely to be a cleavable targeting
sequence. Control western blots, using Tic22, localized to
the intermembrane space, and two outer envelope pro-
teins (atToc159 and atToc33) showed that the protease
behaved as expected in these experiments.
Chloroplast damage, observed by electron microscopy,
is evident in sfr2
The localization of the SFR2 protein to the chloroplast outer
envelope suggested a possible role in the protection of the
chloroplast. In order to investigate this possibility, trans-
mission electron microscopy was used to investigate cellu-
lar damage in sfr2. Comparison of cold-acclimated (10-day
(iii) (ii)
Figure 3. Complementation test of the Arabidopsis sfr2.i2 mutant using the
engineered SFR2
gene that has had its b-glucosidase activity abolished.
(a) Photograph showing the post-freezing phenotypes of: (i) wild-type plants,
(ii) sfr2.i2 plants transformed with the wild-type SFR2, (iii) sfr2.i2 plants and
(iv) sfr2.i2 plants transformed with the G267E SFR2 gene.
(b) Immunoblot demonstrating the expression of the wild-type or engineered
form of SFR2 protein in the indicated plants. The labelling corresponds to that
of the photograph.
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cold treatment) plants showed no differences between sfr2
and wild-type plants (Fig. 6a,b). Material collected from
plants 3 h after their removal from freezing conditions
showed clear differences between wild-type and sfr2 plants.
Cells of wild-type plants were not different in appearance to
those before freezing (Fig. 6e), and chloroplasts had a nor-
mal appearance (Fig. 6f). The cells of sfr2, however, were
clearly damaged. The tonoplast appeared disrupted, and
chloroplasts were present throughout the cell volume
(Fig. 6g). Examination of the chloroplasts showed that they
were damaged: although thylakoid membranes could be
seen, they were loosely packed within the chloroplast
(Fig. 6h). It is clear that 3 h after removal from freezing
conditions, cells of sfr2 are badly damaged, as are the
chloroplast of these cells. In order to obtain a clearer picture
of the nature of the damage in sfr2, further examinations
were carried out on plants just 1 h after removal from
freezing conditions. Many cells were already similar in
appearance to those observed after 3 h, with damaged
chloroplasts dispersed throughout the cell volume. How-
ever, there were cells (approximately 25%) that clearly had
damaged chloroplasts (Fig. 6d), but still appeared to have an
intact tonoplast that restricted the chloroplasts to the
periphery of the cell (Fig. 6c). This suggests that the earliest
signs of damage are to the chloroplast, and that chloroplast
damage is not a product of the release of the vacuole
(a) (b)
(c) (d)
(e) (f)
Figure 4. Localization of SFR2 to the chloroplast
Untagged (empty vector) GFP is clearly localized
in the cytosol, and is not associated with the
chloroplast (a and b). A full-length SFR2 con-
struct tagged with EGFP is specifically associated
with the chloroplast (c), where it is localized to
the chloroplast envelope (d). A construct encod-
ing the first 31 amino acids of SFR2 fused to GFP
shows the same localization (e), and is clearly
also targeted to the chloroplast envelope (f). All
of the displayed images were the result of
transformation of sfr2.i2 plants. GFP fluores-
cence is shown as green and chlorophyll auto-
fluorescence is shown as red. Scale bars: 10 lm.
SFR2 and freezing tolerance in Arabidopsis 739
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contents following tonoplast damage. Tonoplast disruption
may be a result of direct contact with the compromised
chloroplast envelope, or occur through the release of toxic
components from the damaged chloroplast. These early
indications of chloroplast damage suggest a putative role for
the SFR2 protein in the protection of this organelle.
This work has established that SFR2 is widespread
throughout higher plants. The genome sequences of Ara-
bidopsis, and rice (Opassiri et al., 2006), encode a single
SFR2 gene, which in each case is uniquely divergent from
other members of the large b-glucosidase gene family
within that species. This suggests that the SFR2 gene has a
different evolutionary origin to other members of the
b-glucosidase gene family, and that the gene arose before
the divergence of higher plant species. This view is further
supported by other evidence from the Arabidopsis genome
sequence, in that the position of the introns within AtSFR2
are different to those of other members of the gene family
(Xu et al., 2004). Although genes with the potential to pro-
duce proteins with weak similarity to SFR2 were found to be
present in bacteria, and in some simple plant species, these
did not appear to be true orthologues (Fig. 2). The lack of
homologous proteins encoded by cyanobacterial and sim-
ple plant genomes suggests the evolutionary origin of SFR2,
despite its chloroplast localization, is unlikely to be related
to the endosymbiotic cyanobacterium, from which chlo-
roplasts originated. Several EST clones from Physcomitrella
patens appear to encode proteins with high homology to
SFR2 (data not shown), suggesting that the SFR2 gene is
present in bryophytes as well as vascular plants. As these
lineages diverged >400 million years ago (Gifford and Fos-
ter, 1989), it seems likely that the SFR2 gene appeared early
in the emergence of land plants, and perhaps has a role
specific to terrestrial life.
Both GFP microscopy and antibody studies confirm
earlier proteomics-based evidence that the SFR2 protein is
targeted to the chloroplast envelope. The GFP localization
pattern is very similar to that observed for AtOEP7 (Lee et al.,
2001) and CHUP1 (Oikawa et al., 2003), which have both
previously been established as outer envelope proteins.
Targeting of proteins to the chloroplast outer envelope by a
non-cleavable hydrophobic N-terminal region, similar to a
signal peptide, has already been demonstrated (Be
dard and
Jarvis, 2008; Hofmann and Theg, 2005; Li and Chen, 1996).
As is the case with AtOEP7 and CHUP1, the N-terminal
hydrophobic section of the SFR2 protein is sufficient to
target GFP to the chloroplast envelope system. Targeting of
AtOEP7 to the chloroplast outer envelope requires a 35
amino acid region comprising the N-terminal hydrophobic
transmembrane domain (TMD), flanked by an adjacent
charged region (three of six residues are charged) immedi-
ately downstream of this (Be
dard and Jarvis, 2008; Lee et al.,
2001). Analysis of the N-terminal regions of the SFR2
proteins identified in this work (Fig. S3) revealed a very
similar pattern. A predicted N-terminal TMD is immediately
followed by a region rich in charged residues in each case,
suggesting a similar targeting mechanism (one that does
not involve proteolytic cleavage) for Arabidopsis SFR2 and
its homologues. The identification of a putative P. sativum
SFR2 homolog in the chloroplast outer envelope membrane
fraction (Schleiff et al., 2003) supports the notion that the
various SFR2 homologues from other species are also
targeted to this location. The similarity in size of the
endogenous SFR2 protein to the in vitro-translated product
(Fig. 5) is consistent with the hypothesis that a non-proces-
sive targeting mechanism is utilized, although the small size
of the predicted cleavage product means this evidence
should be viewed cautiously.
The possible membrane topology of AtSFR2 was exam-
ined using several topology prediction programmes avail-
able on the internet, including TMHMM (http://www.cbs.
dtu.dk/services/TMHMM-2.0/). The consensus prediction
was two TMDs, near each terminus at positions 4–26 and
448–470 in AtSFR2 (a protein of 622 residues). The most
likely orientation of SFR2 in the outer envelope membrane is
with the N and C termini projecting into the intermembrane
Figure 5. Western blot analysis confirmed that SFR2 is present in proteins
isolated from purified chloroplasts, and shows that thermolysin pre-treatment
of intact organelles produces a protected fragment with a mobility equivalent
to 45 kDa.
In vitro-translated, radiolabelled SFR2 (IVT SFR2) is of the same size as that
present in isolated chloroplasts, but is totally degraded by thermolysin
treatment. Control experiments using the same chloroplast preparation show
that atToc159 and atToc33, proteins that are both exposed to the cytosol, are
degraded by thermolysin as expected, whereas Tic22-IV, a protein located in
the intermembrane space, is protected from digestion. Key: +, treatment with
thermolysin (Th) for a standard period of 60 min; ), control.
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Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 734–745
space (where the C-terminus would be protected from
thermolysin), and with the bulk of the protein exposed to
the cytosolic side of the membrane (including the highly
conserved sequences required for catalytic activity of b-
glycosidases). Such an ‘N
orientation would be consistent
with that seen for other proteins targeted to the outer
membrane by a non-cleavable N-terminal targeting signal
(Hofmann and Theg, 2005). This orientation, together with
the predicted location of TMDs, suggests that 175 residues
(20 kDa) at the C-terminus should be protected from
thermolysin treatment, which is a considerably smaller
fragment than that observed experimentally (45 kDa,
Fig. 5); this suggests that access of the thermolysin to
SFR2 may be hindered by other factors, such as protein–
protein interactions or membrane association. Nonetheless,
the partial sensitivity of the SFR2 protein in these protease
experiments clearly demonstrates localization in the outer
envelope membrane, as thermolysin is unable to penetrate
to more internal locations, such as the intermembrane space
(Cline et al., 1984).
(a) (b)
(c) (d)
(e) (f)
(g) (h)
Figure 6. Post-freezing cellular damage in sfr2
revealed using the transmission electron micro-
Following cold acclimation, no differences are
apparent between wild-type (a) and sfr2 (b)
plants. After freezing followed by 3 h of recovery
no damage is apparent in wild-type plants (e),
where the chloroplasts are intact (f). Cells from
sfr2 plants, however, are clearly damaged: the
tonoplast is disrupted and the chloroplsts are
spread throughout the cell (g). Chloroplasts are
expanded and clearly damaged (h). In sfr2 plants
observed after 1 h of recovery from freezing, the
tonoplast appears to be intact, as the chloro-
plasts are maintained at the periphery of the cell
(c), even though chloroplasts are already dam-
aged (d). Scale bars: a, b, c, e and g, 5 lm; d, f and
h, 1 lm.
SFR2 and freezing tolerance in Arabidopsis 741
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Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 734–745
The ability of SFR2 homologues from freezing (and cold-)
sensitive monocotyledonous and dicotyledonous species to
complement the sfr2 mutation demonstrates that these
genes have, at least to a degree, the same functional activity
as the Arabidopsis gene. Either these SFR2 homologues
have a normal function that is distinct from their effect in
protecting Arabidopsis from freezing, or these genes have
the same function but a phenotype is only manifest after
freezing. Freezing sensitive species presumably lack other
prerequisites for freeze tolerance, making the protection
offered by SFR2 insufficient to impart freezing tolerance. Leaf
order dependent freezing sensitivity, whereby young leaves
and meristematic tissues have greater freezing tolerance
than more mature ones (Takagi et al., 2003), is maintained in
the sfr2 mutant. It appears that SFR2 provides protection
which acts in addition to that imparted by other mechanisms.
The removal of this protection reduces the level of freezing
tolerance in both younger and mature leaves, but not to the
same level, and the differential sensitivity of young and
mature tissue is preserved. The localization of the SFR2
protein in the chloroplast outer membrane (Figs. 4 and 5) and
the demonstration of freezing induced chloroplast damage
(Fig. 6) together suggest a role for the protein in the
protection of this organelle. Previous observations that
SFR2 expression occurs predominately in green tissues
(Thorlby et al., 2004) are also supportive of this notion.
Plants exposed to excess light are known to produce
reactive oxygen species (ROS) that can irreversibly damage
components of the chloroplast (Foyer et al., 1994): a process
enhanced by a number of environmental stresses, including
growth at low temperature (Wise and Naylor, 1987). Possible
roles for b-glucosidases in preventing damage caused by
ROS have been suggested. For example, the release of an
anti-oxidant molecule from an inactive precursor by a
b-glucosidase has been described previously (Chong et al.,
2002), as has the accumulation of free-radical scavenging
flavonoid glycosides at the chloroplast envelope (Agati et al.,
2007). A possible role for SFR2, therefore, is the protection of
the chloroplast from damage caused by ROS. The lack of a
phenotype associated with the growth of sfr2 at low temper-
ature (during cold acclimation) suggests this is not the case. If
SFR2 does have such a role, it would be expected that the sfr2
mutant would be sensitive to other treatments known to
result in ROS-induced damage. However, extensive attempts
to discover additional phenotypes associated with the sfr2
mutation have not been successful. These have included
(data not shown) screening plants for the effects of high-light,
drought, salt and osmotic stress, as well as of paraquat, a
well-known inducer of oxidative stress. We also investigated
the level of lipid peroxidation, a marker for ROS, which is
known to increase in response to many abiotic stresses. This
was investigated after cold acclimation, both during and after
freezing, and again no differences were detected between
wild-type and mutant plants (data not shown).
Thus, despite the localization of the SFR2 protein to the
chloroplast outer envelope membrane, and the clear dam-
age to chloroplasts following a freezing episode, the role of
SFR2 in freeze protection remains unresolved. The inability
of the engineered version of SFR2 (which has an altered
active site motif) to complement the freezing sensitivity of
the mutant (Fig. 3) suggests that b-glucosidase activity is
necessary for freeze protection. The lack of a phenotype
when sfr2 plants were challenged with other abiotic
stresses, as well as the inability of biochemical markers for
ROS damage to distinguish between wild-type and the
mutant, suggests that the function of the SFR2 protein is not
directly related to oxidative stress protection.
The SFR2 protein seems more likely to be involved in
ameliorating the damage caused by a different aspect of
freezing sensitivity. Possibilities involve the modification of
the chloroplast envelope to make it more resistant to
freezing, or the processing of metabolites, either at the
chloroplast membrane surface or while they pass through
the membrane, which are involved in freezing tolerance. The
specific nature of the freeze protection associated with SFR2
function remains to be uncovered. Localization of the
protein in the chloroplast, and the clear damage to this
organelle in the mutant following freezing treatment, sug-
gests that targeted metabolomic profiling may be an effec-
tive means of identifying key changes associated with the
mutation and its phenotype. The presence of the gene, and
its functional conservation, in a wide range of higher plant
species suggests that the gene may have a more funda-
mental role than freeze protection.
Experimental procedures
Plant material and growth conditions
Arabidopsis thaliana sfr2-1 and sfr2-i2 were grown as previously
described (Thorlby et al., 2004). G. max was kindly provided
by Dr Edwards (University of Durham, UK), O. sativa was kindly
provided by Dr Chantereau (CIRAD, France) and P. taeda was gen-
erously provided by Dr Campbell (University of Toronto, Canada).
Plant growth conditions and freeze testing was carried out as
previously described (Thorlby et al., 2004). For plant transforma-
tion, plasmid derived from pB7WG2 and pCAMBIA were transferred
to Agrobacterium tumefaciens GV3101/pM90 by electroporation.
Plants were transformed by the floral-dip method (Clough and Bent,
1998) and transformants were selected on compost by spraying
with a 250 mg l
solution of the herbicide Challenge 60 (AgrEvo;
Bayer CropScience, http://www.bayercropscience.com) or by
growth on plates containing an appropriate antibiotic.
Cloning of GFP constructs
The coding sequence of the full-length SFR2 gene was amplified
using the primers 5¢-CACCATGGAATTATTCGCATTGTTA-3 ¢ and
5¢-GTCAAAGGGTGAGGCTAAAG-3¢, and the N-terminal 93-bp
fragment was amplified using the same forward primer and
742 Nicolas Fourrier et al.
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Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 734–745
5¢-GCGACGGAAACGAGAGTAG-3¢. The PCR products were
cloned into the pENTR/D-TOPO
vector (Invitrogen, http://www.
invitrogen.com), from where they were transferred to an Agrobac-
terium binary vector pK7FWG2 (Karimi et al., 2005) using Gateway
LR Clonase II Enzyme Mix (Invitrogen). To assess the localization of
GFP, the coding sequence of GFP from pK7FWG2 (Karimi et al.,
2005) was cloned into pB7WG2 (Karimi et al., 2005), where GFP was
expressed under the control of the 35S promoter.
In order to observe fluorescence, segments of transformed leaves
(0.5 cm
) were cut using a razor blade, and were mounted in water
for imaging. Fluorescence was analysed using a Bio-Rad radiance
2100 confocal microscope (Bio-Rad, http://www.bio-rad.com) using
the excitation from a 488-nm Argon laser and from a 543-nm HeNe
laser. GFP fluorescence was observed using a 560-nm dichroic filter
and a 485–545-nm bandpass emission filter. Chlorophyll autofluo-
rescence was observed using a 560-nm dichroic mirror and a 600-
nm long-pass emission filter. Images were generated using the
Kalman frame averaging filter on four accumulations. Images were
processed using P
CS2 software (Adobe, http://www.
Electron microscopy
Freezing tests were carried out as previously described (Thorlby
et al., 2004). Following cold acclimation leaves were collected for
pre-freeze observations, and the plants were then transferred to a
freezer. Following removal from freezing conditions, plants were
allowed to recover at room temperature (21C) in the dark. Leaves
were collected 1 and 3 h after removal from the freezing apparatus.
Leaf samples were collected from three independent plants for each
time point and genotype.
Leaf samples (1 mm
) were fixed in 3% glutaraldehyde in 0.1
phosphate buffer, pH 7.2, for 1 h. Samples were washed in buffer
(twice), and were then fixed in 1% aqueous osmium tetroxide for
1 h. They were then washed with distilled water (twice), and then
dehydrated through an ethanol series.
Samples were infiltrated with 50:50 absolute alcohol : Spurr resin
overnight, and were then infiltrated in pure resin for 6 h, before
being transferred to fresh resin, and then polymerized overnight at
60C. Sections approximately 90-nm thick were collected and post-
stained in 1% alcoholic uranyl acetate for 20 min, followed by
Reynold’s lead citrate for 5 min, and were examined in a Hitachi
H7000 TEM at 75 kV.
Generation of full-length SFR2 homologues
Total RNA from S. esculentum, G. max, O. sativa, Z. mays and
A. thaliana was extracted from 100 mg of fresh leaves using the
Plant RNA easy kit (Qiagen, http://www.qiagen.com) following the
manufacturer’s recommendations. Total RNA from P. taeda was
extracted from 2 to 3 g of pine needles according to the method
described by Chang et al. (1993).
The Thermoscript RNase H-reverse transcriptase kit (Invitrogen)
was used, according to the manufacturer’s instructions, utilizing
500 ng of total RNA, to produce cDNA. Freshly synthesized cDNA
was used as the template in PCR reactions to amplify full-length
SFR2-like cDNAs.
For O. sativa, Z. mays and G. max, primers were designed in the
5¢ untranslated region (UTR) and the 3¢ UTR of the SFR2-like
sequences obtained after BLAST searches against the AtSFR2
protein sequence: for O. sativa the information was obtained from
a sequenced BAC clone (AC13590), and for Z. mays and G. max
from two EST clones (BM381643 and BI992660, and AW75621 and
AI966213, respectively) covering the 5¢ and 3¢ ends of the gene.
The isolation of full-length SFR2 -like sequence from S. esculen-
tum and P. taeda was performed using the 5 ¢/3¢ RACE strategy
(Frohman et al., 1988), following the instructions of the 5¢/3¢ RACE
kit (Roche, http://www.roche.com).
Sequence information obtained from EST clones covering the
3¢ regions of S. esculentum (AW932205) and P. taeda (BG319035)
were used to design race primers to amplify the 5¢ ends of the genes.
New primers designed using the 5¢ and 3¢ ends then allowed the
amplification of full-length clones. Details of all primers used are
available on request.
All full-length SFR2-like sequences were amplified with either Pfu
polymerase (Promega, http://www.promega.com) or KoD Hifi DNA
polymerase (Novagen, http://www.merckbiosciences.co.uk/html/
NVG/home.html). PCR products were cloned into pGEM-T easy
vector (Promega) and were then sequenced.
For complementation experiments, OsSFR2 cDNA was first
transferred to pENTR4 (Invitrogen) and then transferred into a
GATEWAY plant expression vector pB7WG2 (Karimi et al., 2005)
using the Gateway
LR Clonase Enzyme Mix (Invitrogen). PtSFR2
and GmSFR2 cDNAs were transf erred, by restriction digestion and
ligation, to pCAMBIA 1305.1 (CAMBIA, http://www.cambia.org).
Site-directed mutagenesis
In order to introduce a mutation in the AtSFR2 cDNA, four primers
were designed, two of which are flanking, and two of which cover
and introduce the point mutation into the amplified material. Two
independent PCRs were performed using Pfu polymerase (Pro-
mega) to generate the two halves of the final product. The template
used in both PCRs was a plasmid containing the full-length AtSFR2
cDNA. Primers for the first PCR were 5¢-CATTTAATGGACC-
GCTAAAG-3¢, and for the second PCR were 5¢-GTGAAGATA-
TATTCGCATTGTT-3¢. PCR products were gel purified using a Gel
Purification Kit (Qiagen). Products were combined in a third PCR
using two flanking oligonucleotides: 5¢-CTCTAAAAATGTTGCCGC-
GT-3¢ and 5¢-CACACTGCTTCCTGTCCGTA-3¢. The final product
AtSFR2.G267E was then cloned into the pGEM-T easy vector, and
was sequenced before transfer to an Agrobacterium binary vector.
Protein extraction and immunoblots
Total protein from 5-week-old plants was isolated using the method
described by Martinez-Garcia et al. (1999). Equal quantities (20 lg)
of protein were loaded per lane and separated by SDS-PAGE before
transfer onto polyvinylidene difluoride membrane by electro-blot
transfer. Chloroplasts isolation and thermolysin treatments were as
described previously (Aronsson and Jarvis, 2002). Transcription/
translation was performed in a coupled system using rabbit retic-
ulocyte lysate and T7 RNA polymerase (TNT T7 Quick for PCR DNA;
Promega) containing [
S]methionine (Aronsson and Jarvis, 2002).
Anti-SFR2 antibody, raised against a peptide comprising amino
acids 514–622, was used as described by Thorlby et al. (2004). Anti-
atToc159 was as described by Bauer et al. (2000), and was raised
against the A domain of the protein. Anti-atToc33 was raised against
a C-terminal peptide, and was used as described by Aronsson et al.
(2003). Anti-Tic22 polyclonal antibody was raised against a
synthetic atTic22-IV-specific peptide (CIERELSKYTRASRGD) by
Eurogentec (http://www.eurogentec.com).
SFR2 and freezing tolerance in Arabidopsis 743
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Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 55, 734–745
Bioinformatics and phylogenetic analysis
Amino acid sequences were aligned by C
X (Thompson et al.,
1997), and were manually inspected for obvious irregularities. All
positions with one or more gaps in the alignment were removed
from the dataset to be used for phylogenetic analysis, as well as
regions flanking gaps where alignment was unreliable. This
resulted in a dataset containing 230 amino acid positions. Phylo-
genetic analyses were carried out using PAUP*4.10b (Swofford,
2003). Neighbour-joining trees were constructed using the BioNJ
method using total-character distances. Bootstrap analyses with
1000 replicates were used to assess the relative support for the
tree topology.
Sequences used in the bioinformatic analysis are the most similar
procaryotic b-glycosidases: Thermosphaera aggregans (AAD43138)
and Thermosphaera sp. (CAA94187), A. thaliana SFR2, and
sequences from O. sativa SFR2 homologue (AJ491323), G. max
SFR2 homologue (AM238658), S. esculentum SFR2 homologue
(AM238659), Z. mays SFR2 homologue (AM238660) and P. taeda
SFR2 homologue (AM238661).
Thanks are due to Tom Ford for assistance and advice with electron
microscopy. We are grateful to Felix Kessler for the generous gift of
the Anti-Toc159 antibody.
Supporting Information
Additional supporting information may be found in the online
version of this article.
Figure S1. Post-freezing phenotype Arabidopsis sfr2.i2 plants inde-
pendently transformed with cDNAs encoding PtSFR2 (A), GmSFR2
(B), OsSFR2 (C) and AtSFR2 (D).
Figure S2. Leaf order dependent freezing sensitivity is preserved in
Figure S3. Comparison of the N-terminal region of AtOEP7 with the
equivalent regions of AtSFR2, and the SFR2-like sequences
described in this work.
Please note: Blackwell publishing are not responsible for the
content or functionality of any supporting materials supplied by
the authors. Any queries (other than missing material) should be
directed to the corresponding author for the article.
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SFR2 and freezing tolerance in Arabidopsis 745
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    • "Stress-induced TAG accumulation has also been observed in Arabidopsis during freezing (Moellering and Benning 2011 ;). Studies on Arabidopsis SENSITIVE TO FREEZING 2 ( SFR2 ) (), a gene encoding a protein annotated as a glycosyl hydrolase family 1 protein at the outer chloroplast envelope membrane (Fourrier et al. 2008 ; Thorlby et al. 2004 ), suggested that SFR2 likely is the GGGT in plants and participates in the protection of chloroplast under freezing stress due to its activity causing the formation of oligogalactolipids and DAG by processive transfer of galactosyl moieties from MGDG onto an galactolipid acceptor ( ). A more recent study confi rmed that SFR2 acts solely as a glycosyltransferase rather than a glycosyl hydrolase and provided insights into its reaction mechanism through structure function studies assisted by a structural homology model (Roston et al. 2014 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Plant and algal oils are some of the most energy-dense renewable compounds provided by nature. Triacylglycerols (TAGs) are the major constituent of plant oils, which can be converted into fatty acid methyl esters commonly known as biodiesel. As one of the most efficient producers of TAGs, photosynthetic microalgae have attracted substantial interest for renewable fuel production. Currently, the big challenge of microalgae based TAGs for biofuels is their high cost compared to fossil fuels. A conundrum is that microalgae accumulate large amounts of TAGs only during stress conditions such as nutrient deprivation and temperature stress, which inevitably will inhibit growth. Thus, a better understanding of why and how microalgae induce TAG biosynthesis under stress conditions would allow the development of engineered microalgae with increased TAG production during conditions optimal for growth. Land plants also synthesize TAGs during stresses and we will compare new findings on environmental stress-induced TAG accumulation in plants and microalgae especially in the well-characterized model alga Chlamydomonas reinhardtii and a biotechnologically relevant genus Nannochloropsis.
    Full-text · Article · Mar 2016 · The Plant Journal
    • "This is consistent with a previous study showing that isolated plastidial envelops of Chlamydomonas do not synthesize oligogalactolipids including TGDG (Mendiola-Morgenthaler et al. 1985). Furthermore, phylogenetic analysis has indicated that SFR2 orthologs are restricted to land plant species and not found in bacteria or simple algae (Fourrier et al. 2008). In addition, the expression of the Chlamydomonas gene encoding the protein with the highest sequence similarity to SFR2 was not found to be upregulated upon heat stress in our transcriptomic study. "
    [Show abstract] [Hide abstract] ABSTRACT: Studying how photosynthetic cells modify membrane lipids in response to heat stress is important to understand how plants and microalgae adapt to daily fluctuations in temperature and to investigate new lipid pathways. Here we investigate changes occurring in lipid molecular species and lipid metabolism genes during early response to heat stress in the model photosynthetic microorganism Chlamydomonas reinhardtii. Lipid molecular species analyses revealed that, after 60 min at 42°C, a strong decrease in specific polyunsaturated membrane lipids were observed together with an increase in polyunsaturated triacylglycerols (TAGs) and diacylglycerols (DAGs). The fact that decrease in the major chloroplastic monogalactosyldiacylglycerol MGDG sn1-18:3/sn2-16:4 was mirrored by an accumulation of DAG sn1-18:3/sn2-16:4 and TAG sn1-18:3/sn2-16:4/sn3-18:3 indicated that newly accumulated TAGs were formed via direct conversion of MGDGs to DAGs then TAGs. Lipidomic analyses showed that the third fatty acid of a TAG likely originated from a phosphatidylethanolamine or a DGTS betaine lipid species. Candidate genes for this TAG synthesis pathway were provided through comparative transcriptomic analysis and included a phospholipase A2 homolog and the diacylglycerol acyltransferase DGTT1. This study gives insights into lipidomic and transcriptomic responses underlying changes in membrane lipids during heat stress and reveals an alternative route for triacylglycerol synthesis.
    Full-text · Article · Oct 2015
    • "Craterostigma plantagineum presumably also contains a gene orthologous to AtDGD2, but no corresponding CpDGD2 sequence was retrieved. In Arabidopsis, SFR2, DGD1 and DGD2 are localized to the chloroplast envelope, where they have access to only a low proportion of MGDG while most of the galactolipids are in the thylakoids (Froehlich et al., 2001; Fourrier et al., 2008). Therefore, it is conceivable that only a limited proportion of MGDG can be converted into DGDG and oligogalactolipids by the DGD1/ DGD2 and SFR2 pathways. "
    [Show abstract] [Hide abstract] ABSTRACT: Dehydration leads to different physiological and biochemical responses in plants. We analyzed the lipid composition and the expression of genes involved in lipid biosynthesis in the desiccation-tolerant plant Craterostigma plantagineum. A comparative approach was carried out with Lindernia brevidens (desiccation tolerant), and two desiccation-sensitive species, L. subracemosa and A. thaliana. In C. plantagineum the total lipid content remained constant while the lipid composition underwent major changes during desiccation. The most prominent change was the removal of monogalactosyldiacylglycerol (MGDG) from the thylakoids. Analysis of molecular species composition revealed that around 50% of 36:x (number of carbons in the acyl chains: number of double bonds) MGDG was hydrolyzed and diacylglycerol (DAG) used for phospholipid synthesis, while another MGDG fraction was converted into digalactosyldiacylglycerol via the DGD1/DGD2 pathway and subsequently into oligogalactolipids by SFR2. 36:x-DAG was also employed for the synthesis of triacylglycerol. Phosphatidic acid (PA) increased in C. plantagineum, L. brevidens, and L. subracemosa, in agreement with a role of PA as an intermediate of lipid turnover and of phospholipase D in signalling during desiccation. 34:x-DAG, presumably derived from de novo assembly, was converted into phosphatidylinositol (PI) in C. plantagineum and L. brevidens, but not in desiccation-sensitive plants, suggesting that PI is involved in acquisition of desiccation tolerance. The accumulation of oligogalactolipids and PI in the chloroplast and extraplastidial membranes, respectively, increases the concentration of hydroxyl groups and enhances the ratio of bilayer to nonbilayer forming lipids, thus contributing to protein and membrane stabilization. This article is protected by copyright. All rights reserved.
    Full-text · Article · May 2013
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