Plant Cell Physiol. 49(9): 1390–1395 (2008)
doi:10.1093/pcp/pcn103, available online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Short Communication
A Unique Caleosin in Oil Bodies of Lily Pollen
Pei-Luen Jiang 1, Guang-Yuh Jauh 2, Co-Shing Wang
1
and Jason T.C. Tzen
1, 3,
*
1
Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, Taiwan
2
Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan
3
Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan
In view of the recent isolation of stable oil bodies as well
as a unique oleosin from lily pollen, this study examined
whether other minor proteins were present in this lipid-storage
organelle. Immunological cross-recognition using antibodies
against three minor oil-body proteins from sesame suggested
that a putative caleosin was specifically detected in the oilbody fraction of pollen extract. A cDNA fragment encoding
this putative pollen caleosin, obtained by PCR cloning, was
confirmed by immunodetection and MALDI-MS analyses of
the recombinant protein over-expressed in Escherichia coli
and the native form. Caleosin in lily pollen oil bodies seemed
to be a unique isoform distinct from that in lily seed oil bodies.
Keywords: Caleosin — Lily (Lilium longiflorum Thunb.) —
Oil bodies — Oleosin — Pollen.
Abbreviations: PL(s),
phospholipid(s);
TAG(s),
triacylglycerol(s).
The nucleotide sequence reported in this paper has
been submitted to the GenBank Data Bank with accession
number EF015588.
Eukaryotes contain high amounts of neutral lipids,
mainly triacylglycerols (TAGs) and sterol esters, in intraand extra-cellular granules (Davis and Vance 1996). In
plant seeds, the neutral storage lipids are confined to
discrete spherical organelles called oil bodies (Yatsu and
Jacks 1972, Huang 1996). An oil body is proposed to
comprise a TAG matrix covered by a layer of phospholipids
(PLs) embedded with some unique proteins. The most
thoroughly characterized seed oil-body protein is oleosin,
an alkaline protein ranging in size from 15 to 30 kDa
(Murphy 2001).
In the past decade, two additional classes of oil body
proteins, caleosin and steroleosin, have been identified in
seeds (Chen et al. 1999, Næsted et al. 2000, Lin et al. 2002,
Lin and Tzen 2004). Caleosin, with a calcium-binding motif
and several potential phosphorylation sites, may be
regulated for some biological functions related to the
synthesis or degradation of oil bodies (Frandsen et al.
2001, Poxleitner et al. 2006). It has also been shown to
possess a peroxygenase activity and is thought to be related
to phytooxylipin metabolism (Hanano et al. 2006).
Steroleosin comprises a sterol-binding dehydrogenase that
belongs to a super-family of pre-signal proteins involved in
signal transduction (Tzen et al. 2003).
Similar to seeds, pollen grains contain a large number
of intracellular oil bodies that serve as energy reserves for
subsequent germination (Piffanelli et al. 1998). Taking
advantage of large pollen grains in lily flower, we
successfully isolated stable pollen oil bodies that contained
a TAG matrix surrounded by a monolayer of PLs
embedded with proteins (Jiang et al. 2007). The surface
proteins that maintained the integrity of these organelles via
electronegative repulsion and steric hindrance comprised an
abundant oleosin and possibly some minor proteins of
higher molecular mass. Whether minor proteins in pollen oil
bodies are similar to those in seed oil bodies has not been
addressed.
In this study, we intended to investigate if any other
proteins were co-existent with the unique and abundant
oleosin in lily pollen oil bodies. Proteins extracted from oil
bodies of lily pollen were screened by immunological crossrecognition using antibodies against three minor proteins of
sesame oil bodies, and then subjected to mass spectrometric
analysis. A putative caleosin was identified, cloned, and
characterized in pollen oil bodies as a unique isoform
distinct from that present in seed oil bodies of lily.
Comparable to sesame seed oil bodies, several minor
proteins besides the abundant oleosin were observed in lily
pollen oil bodies (Fig. 1, left panel). To examine if
analogous minor proteins were present in these two types
of oil bodies, proteins extracted from pollen oil bodies were
subjected to immunological cross-recognition using antibodies against three minor proteins in sesame oil bodies,
caleosin (27 kDa), steroleosin-A (39 kDa), and steroleosin-B
(41 kDa). Immunodetection indicated that a putative
caleosin of 28 kDa was exclusively present in the oil-body
fraction of pollen extract (Fig. 1) while the pre-immune
antibodies did not recognize any protein bands. Similar to
*Corresponding author: E-mail, [email protected]: Fax, þ886-4-22853527.
1390
lily
pollen
sesame
seed
lily
pollen
sesame
seed
1391
lily
pollen
sesame
seed
lily
pollen
sesame
seed
lily
pollen
sesame
seed
lily
pollen
sesame
seed
lily
pollen
sesame
seed
Caleosin in lily pollen oil body
kDa
41
41
41
39
39
39
27
27
27
17
15
SDS-PAGE
Ab-caleosin
Ab-steroleosin-A
Ab-steroleosin-B
Ab-caleosin
pre-immune
Ab-steroleosin-A
pre-immune
Ab-steroleosin-B
pre-immune
Fig. 1 SDS-PAGE and Western blots of proteins in oil bodies of sesame seed and lily pollen. Proteins extracted from oil bodies of sesame
seed and lily pollen were resolved by SDS-PAGE and subjected to immunodetection using antibodies or pre-immune antibodies against
sesame caleosin (27 kDa), steroleosin-A (39 kDa), and steroleosin-B (41 kDa), respectively. The molecular masses of sesame seed oil-body
proteins including two oleosin isoforms of 15 and 17 kDa are indicated on the left.
the known oleosin of 18 kDa, this putative caleosin was not
detected in other tissues, including root, stem, leaf, tepal,
ovary, style, filament, and stigma (data not shown). The
putative pollen caleosin, after trypsin digestion, produced a
fragment GAFDGSLFER, which matched a tryptic fragment of the theoretical rice caleosin (accession No.
AAN05334) in a mass spectrometric analysis.
A full-length cDNA fragment (accession No.
EF015588) encoding the putative pollen caleosin was
obtained by PCR cloning. It comprises 986 nucleotides,
including a 50 -untranslated region of 50 nucleotides, an
open reading frame of 720 nucleotides, and a 30 -untranslated region of 216 nucleotides. The deduced polypeptide of
239 amino acid residues with a molecular mass of 26,678 Da
is homologous to caleosins from diverse species. Sequence
alignment indicates that pollen caleosin comprises three
structural domains including an N-terminal hydrophilic
calcium-binding domain, a central hydrophobic oil-body
anchoring domain, and a C-terminal hydrophilic phosphorylation domain (Fig. 2A). In comparison with known seed
calesoins of two monocot and two dicot species, all three
structural domains are highly conservative, particularly in
the Caþ2 binding motif and the central hydrophobic
domain. As expected, a proline-knot motif presumably
responsible for protein targeting to oil bodies is also present
in the central hydrophobic domain of pollen caleosin. The
10-residue tryptic fragment identified in mass analysis exists
in the C-terminal region of this putative caleosin from lily
pollen oil bodies as well as the theoretical rice 30 kDa
caleosin. Phylogenetic tree analysis suggests that pollen
caleosins might represent a distinct isoform compared with
caleosins found in seed oil bodies (Fig. 2B).
To examine if the caleosin clone truly encoded the
28 kDa caleosin found in pollen oil bodies, it was overexpressed in Escherichia coli as a fusion protein and
subjected to immunodetection. The result indicated that
the recombinant fusion protein of 46 kDa and native pollen
caleosin of 28 kDa could be cross-recognized by antibodies
against sesame caleosin (data not shown). Furthermore,
MALDI-MS analyses showed that tryptic fragments of
both proteins matched to the amino acid sequence deduced
from the DNA sequence of the lily pollen caleosin clone
(Table 1).
To see if the pollen caleosin is different from the seed
caleosin in lily, proteins extracted from oil bodies of lily
seed and pollen were resolved in SDS-PAGE and subjected
to immunodetection (Fig. 3). Compared with the caleosin in
pollen oil bodies, a putative caleosin of slightly higher
molecular mass was detected in seed oil bodies while the
pre-immune antibodies did not recognize any protein
bands. The distinction between lily pollen and seed caleosins
was further supported by MALDI-MS analyses (Table 1).
In contrast with the widespread match of tryptic fragments
in the identification of lily pollen caleosin, no match was
detected for tryptic fragments of the putative caleosin in lily
seed oil bodies. Instead, three tryptic fragments of the
putative lily seed caleosin matched to the amino acid
sequence of caleosin in sesame seed oil bodies. Apparently,
distinct caleosin isoforms were present in the oil bodies of
lily seed and pollen.
1392
Caleosin in lily pollen oil body
N-terminal domain
A
35
35
36
30
67
31
Rice
Barley
Sesame
Soybean
Rice P
lily P
Ca+2 binding motif
99
101
100
94
131
95
Rice
Barley
Sesame
Soybean
Rice P
lily P
C-terminal domain
Central
domain
proline-knot
135
137
136
130
167
131
Rice
Barley
Sesame
Soybean
Rice P
lily P
Rice
Barley
Sesame
Soybean
Rice P
lily P
199
202
201
195
232
196
Rice
Barley
Sesame
Soybean
Rice P
lily P
244
246
245
239
271
239
B
Rice
Barley
Sesame
Soybean
Rice P
Lily P
38.3
35
30
25
20
15
10
Nucleotide Substitutions (× 100)
5
0
Fig. 2 Sequence alignment and phylogenetic tree analysis of the pollen caleosin and known caleosin isoforms from seeds. (A) Sequences
of the lily pollen caleosin, the putative rice pollen caleosin, and four known caleosins in two monocot (rice and barley) and two dicot
(sesame and soybean) seeds were used for alignment. Sequences are aligned according to their three structural domains (N-terminal,
central hydrophobic and C-terminal domains). The amino acid number of the last residue of each domain is listed on the right for each
sequence. Broken lines in the sequences show gaps introduced for the best alignment, and conserved residues are shaded. The positions of
a calcium-binding motif and a proline-knot motif are indicated on tops of their sequences. The partial amino acid sequence identified in
the mass analysis is enclosed. The residues used for designing a pair of degenerate primers are indicated by a right and a left arrow,
respectively. The accession numbers of the aligned caleosin sequences are: rice seed, X89891; barley, AAQ74240; sesame, AF109921;
soybean, AF004809; rice pollen (P), AAN05334; and lily pollen, EF015588. (B) Phylogenetic tree analysis of the six caleosin sequences.
The scale represents branch distance as the number of residue changes between neighbors.
To date, three classes of proteins, oleosin, caleosin and
steroleosin, have been identified in seed oil bodies, and
isoforms are likely to be present in each class within the
same oil bodies (Tzen et al. 2003). In our previous study,
stable oil bodies were successfully isolated from lily pollen
and a unique oleosin isoform was identified in these purified
organelles, in contrast with the two oleosin isoforms
exclusively found in seed oil bodies of diverse angiosperm
species (Jiang et al. 2007). In this study, a distinct isoform of
caleosin was identified in pollen oil bodies in comparison
with the caleosin isoforms universally found in various seed
oil bodies. Although immunological screening using antibodies against sesame steroleosin isoforms did not produce
positive signals, we could not rule out the possibility for the
presence of steroleosin isoforms in the oil bodies of lily
pollen. Further investigations will also be made to see if
pollen oil bodies contain novel minor proteins different
from known seed oil body proteins.
Calcium plays a key role in sexual plant reproduction,
and some calcium-binding proteins in plants, such as
Lily protein
Pollen
caleosin
Recombinant
caleosin
Residues
69–74
136–148
149–153
154–160
199–205
206–213
218–227
1–24
25–41
26–41
75–90
114–129
149–160
154–160
199–205
199–213
218–227
Seed
caleosin
Sequence
(lily pollen caleosin)
AAFFDR
HGSDTESYDTEGR
FEPSK
FDAIFSK
LLYQIGK
DEDGLLHK
GAFDGSLFER
MGSTSDPSPSIITV
AAEAPVTAER
KQNLHLQEQLAK
PYVAR
QNLHLQEQLAKPYVAR
NNDGIVYPWETYQGFR
SQPSWIPSPVLSIHIK
FEPSKFDAIFSK
FDAIFSK
LLYQIGK
LLYQIGKDEDGLLHK
GAFDGSLFER
Residues
Sequence
(sesame seed caleosin)
141–153
166–173
211–218
HGSDSGTYDTEGR
HARTMPDR
DQDGFLSK
calmodulin, calcium-dependent protein kinases and cytoskeletal proteins, have been proposed to modulate downstream processes involved in pollen germination and pollen
tube growth (Moutinho et al. 1998, Snowman et al. 2002,
Golovkin and Reddy 2003, Rato et al. 2004). Caleosin that
is found present in lily pollen oil bodies in this study
provides a potential target for calcium, although its role on
the surface of this lipid storage organelle remains to be
explored. Recently, caleosin was demonstrated to possess a
peroxygenase activity possibly related to phytooxylipin
metabolism, and calcium was found to be crucial for this
activity (Hanano et al. 2006). It remains to be studied if the
peroxygenase activity of caleosin plays a role in the
biosynthesis or degradation of oil bodies.
According to ultrastructural observation, oil bodies in
seed and pollen are likely to undergo different mobilization
routes for their storage TAGs (Tzen et al. 1997, Jiang et al.
2007). In germinating seeds and seedlings, oil bodies would
come into contact and fuse with glyoxysomes, into which
free fatty acids released from TAGs are transported for
lily
pollen
lily
seed
lily
pollen
lily
seed
lily
pollen
1393
lily
seed
Table 1 Fragments of lily pollen caleosin, recombinant
caleosin and seed caleosin identified by MALDI-MS
analyses
Marker
Caleosin in lily pollen oil body
kDa
76
52
38
kDa
31
29
28
24
17
12
SDS-PAGE
Ab-caleosin
Ab-caleosin
pre-immune
Fig. 3 Immunodetection of caleosin in oil bodies of lily seed and
pollen. Proteins extracted from oil bodies of lily seed and pollen
were resolved by SDS-PAGE. A duplicate gel was transferred onto a
nitrocellulose membrane and subjected to immunodetection using
antibodies or pre-immune antibodies against sesame caleosin. The
molecular masses of marker proteins are indicated on the left, and
those of a putative seed caleosin and the pollen caleosin (29 and
28 kDa, respectively) on the right.
b-oxidation (Trelease 1984). In contrast, pollen oil bodies,
individually surrounded by tubular membrane structures,
were encapsulated in the vacuoles after germination and
possibly mediated by the vacuolar digestion pathway
(Bassham et al. 2006). However, the apparent fusion of oil
bodies with vacuoles has also been reported in germinating
maize kernels, and proposed as part of the TAG mobilization (Wang and Huang 1987). Whether both TAG
degradation pathways exist and correspond to different
stages of oil-body mobilization remains to be investigated.
Nevertheless, seed and pollen oil bodies are extremely
similar in their structure, stability, particle size, and
composition. It will be interesting to see if similar oil
bodies could be mobilized in two different routes when
exposed to different environments, i.e., seed or pollen
germination, or to identify distinct constituents (factors) in
these two types of oil bodies responsible for the different
degradation pathways of these lipid storage organelles after
germination.
Materials and Methods
Plant materials
Anthers of lily (Lilium longiflorum Thunb. cv. Snow Queen)
flowers were purchased from a local farmer. Mature pollen was
harvested 2 d after anthesis when the size of lily bud was
approximately 165–170 mm. Tissues dissected from mature lily
plants including root, stem, leaf, tepal, stigma, style, ovary, and
filament were frozen immediately in liquid nitrogen and stored
1394
Caleosin in lily pollen oil body
at 808C until use. Mature sesame and lily seeds were harvested,
air dried, and stored at room temperature.
Purification of oil bodies from lily seed and pollen
Seed oil bodies extracted from sesame and lily were subjected
to further purification using the protocol developed by Tzen et al.
(1997). Mature pollen grains collected by filtration through 30 mm
nylon mesh were homogenized at 48 C in a grinding medium
(8 g pollen per 35 ml) containing 0.6 M sucrose and 10 mM sodium
phosphate buffer, pH 7.5. Oil bodies in the pollen extract
were isolated according to the method described previously
(Jiang et al. 2007).
SDS-PAGE and Western blotting
SDS-PAGE and Western blotting were executed as described
previously (Jiang et al. 2007). Primary antibodies against sesame
seed oil-body proteins (27 kDa caleosin, 39 kDa streoleosin-A, and
41kDa streoleosin-B) or pollen 18 kDa oleosin (Chen et al. 1999,
Lin et al. 2002, Lin and Tzen 2004, Jiang et al. 2007) were used for
immunodetection.
MALDI-MS and MALDI-MS/MS analyses
Protein bands of lily pollen caleosin, recombinant caleosin
and seed caleosin resolved in SDS-PAGE were manually excised
from the gel and subjected to in-gel digestion as described before
(Jiang et al. 2007). Tryptic peptides derived from in-gel digestion of
lily proteins were analyzed by MALDI-MS and MALDI-MS/MS.
The MS/MS data were used to search algorithms against the SwissProt database using Mascot software (Matrix Science Ltd.,
London, UK).
Cloning of a putative cDNA fragment encoding 28 kDa caleosin in
lily pollen oil bodies
Total RNA was isolated from maturing lily pollen and
corresponding cDNA fragments were subsequently synthesized
as described previously (Jiang et al. 2007). To clone the putative
pollen caleosin, two degenerate primers, 50 -ARCAYGBBGCBTT
CTTYGA-3 and 50 -WACCTTCCTTCHKHRTC-30 , were designed
according two conservative regions found in caleosins from
diverse species. The first PCR fragment of approximately 230 bp
was obtained using the two degenerate primers with the pollen
cDNA fragments as templates. To obtain the downstream
sequence, a specific primer, 50 -TGGAAGCGACACCGAAAGC
TAC-30 , was designed according to the sequence obtained from
the first PCR fragment. The second PCR fragment of
approximately 530 bp was obtained by 30 RACE. To obtain the
upstream sequence, a specific primer, 50 -GATAAACAATCC
CGTCATTGTTCC-30 was designed according to the sequence
obtained from the first PCR fragment. The third PCR fragment
of approximately 294 bp was obtained by 50 RACE. The complete
cDNA clone of 986 bp was linked by PCR and sequenced from both
directions. Sequence comparisons were performed with the
GenBank using Blast program (Altschul et al. 1990). Phylogenetic
tree was analyzed using the CLUSTAL program as part of
Megalign (DNASTAR Inc., Madison, WI).
Over-expression of lily pollen caleosin in E. coli
The full-length cDNA clone of lily pollen caleosin was
constructed in the fusion expression vector, pET32a(þ) (Novagen),
using an EcoRI and a XhoI site in the polylinker of the vector. The
recombinant plasmid was used to transform E. coli strain
BL21(DE3). Over-expression of the recombinant fusion protein
was induced by 1 mM IPTG in a bacteriophage T7 RNA
polymerase/promoter system. Four hours after induction, the
E. coli cells were harvested, cracked by sonication in 200 ml
extraction buffer (10 mM Tris–HCl, pH 7.4, 1 mM PMSF, 100 mM
NaCl, 1 mM DTT, and 1 mM EDTA), and then centrifuged at
15,000g for 10 min at 48C. After centrifugation, the supernatant
containing the fusion protein was subjected to further analyses by
immunoassaying and MALDI-MS.
Funding
National Science Council, Taiwan, ROC (NSC 962628-B-005-003-MY3 to JTC Tzen).
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(Received May 15, 2008; Accepted July 13, 2008)