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First published online May 22, 2003; 10.1104/pp.103.020875 Plant Physiology 132:1249-1259 (2003) © 2003 American Society of Plant Biologists Localization of Nonspecific Lipid Transfer Proteins Correlate with Programmed Cell Death Responses during Endosperm Degradation in Euphorbia lagascae Seedlings1Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Box 7080, 750 07 Uppsala, Sweden
When the storage materials have been depleted, the endosperm cells undergo programmed cell death. Very little is known about how the components of the dying cells are recycled and used by the growing seedling. To learn more about endosperm degradation and nutrient recycling, we isolated soluble proteins from the endosperm of Euphorbia lagascae seedlings collected 2, 4, and 6 d after sowing. The protein extracts were subjected to two-dimensional gel electrophoresis. Proteins that increased in amount in the endosperm with time were selected for further analysis with mass spectrometry. We successfully identified 17 proteins, which became more abundant by time during germination. Among these proteins were three E. lagascae lipid transfer proteins (ElLTPs), ElLTP1, ElLTP2, and ElLTP3. Detailed expressional studies were performed on ElLTP1 and ElLTP2. ElLTP1 transcripts were detected in endosperm and cotyledons, whereas ElLTP2 transcripts were only detected in endosperm. Western blots confirmed that ElLTP1 and ElLTP2 accumulate during germination. Immunolocalization experiments showed that ElLTP1 was present in the vessels of the developing cotyledons, and also in the alloplastic space in the endosperm. ElLTP2 formed a concentration gradient in the endosperm, with higher amounts in the inner regions close to the cotyledons, and lesser amounts in the outer regions of the endosperm. On the basis of these data, we propose that ElLTP1 and ElLTP2 are involved in recycling of endosperm lipids, or that they act as protease inhibitors protecting the growing cotyledons from proteases released during programmed cell death.
The endosperm is a tissue that is destined to be degraded, so when the storage materials are depleted, the cells of the endosperm undergo senescence by programmed cell death (PCD; Schmid et al., 1999  ). In senescing tissues, the degree of processing of dead
and dying cells ranges from that apparently limited to the nucleus or nDNA to
complete autolysis. One important route for recycling of proteins is vacuolar
autophagy, which takes place during starvation and PCD
(Reggiori and Klionski, 2002).
In autophagy in yeasts, a portion of the cytoplasm is enclosed by the
autophagosome, which is a double-membraned structure. The autophagosome then
fuses to the vacuole, where the cytoplasmic contents are degraded. The system
is similar in animal cells, although here the lysosome acts as the degrading
compartment. One of the essential components of the autophagic system in
yeasts is a pair of conjugation pathways that attach polypeptide tags to the
lipid phosphatidylethanolamine and the protein APG8
(Ichimura et al., 2000). By an
unknown mechanism, these modifications trigger autophagosome assembly and
fusion of the vesicles with the vacuole.
There is probably a similar system for autophagy in plant tissues, because
the genome of Arabidopsis contain at least 25 APG genes that are homologous to
yeast genes essential for autophagy. Interestingly, leaf senescence was shown
to accelerate in Arabidopsis lacking a functional gene for APG9
(Hanaoka et al., 2002
It is not known whether autophagy or other cellular activities are involved
in the degradation and recycling of cellular components from senescing
endosperm cells. Previously, we observed that transcription of nonspecific
lipid transfer proteins (LTPs) was induced during germination, and we
suggested that LTPs may be involved in the recycling of lipids from senescing
endosperm cells (Edqvist and Farbos,
2002
LTPs are not specified to any certain tissue or organ, instead they have
been found in various tissues throughout the whole plant but in some tissues
only at certain developmental stages. For example, LTPs have been found in
potato (Solanum tuberosum) tubers
(Horvath et al., 2002
To learn more about endosperm degradation and recycling and to elucidate
the role of LTPs in such processes, we decided to follow the development of
the endosperm proteome of Euphorbia lagascae during germination. In
E. lagascae, the endosperm contain high amounts of the epoxidated
fatty acid vernolic acid, which has potential industrial usages in paint,
coating, and lubricant production as an alternative to petroleum derived oils.
Our research interest was originally on enzymes participating in the
metabolism of vernolic acid during seed germination (Edqvist and Farbos,
2002
Endosperm Morphology of Germinating E. lagascae Seedlings
The seed structure of E. lagascae resembles very much that of
castor bean (Ricinus communis), a well-established model organism for
studies on seed germination (Beevers,
1980
To identify proteins involved in endosperm degradation, we extracted
proteins from the endosperm at 2, 4, and 6 d of germination. The protein
extracts were precipitated with TCA and analyzed by two-dimensional gel
electrophoresis (Fig. 2). The
2-, 4-, and 6-d proteomes can easily be told apart by the pattern of proteins
with a molecular mass of approximately 25 kD and pI between 7 and 10. These
proteins are probably mainly oleosins
(Huang, 1996
In the two-dimensional gels, we identified 17 spots, which increased in
size and density during germination (Fig.
2). We cut these spots, indicated in
Figure 2, B, C, and F, from the
gels, digested the proteins with trypsin, extracted the peptides, and finally
sequenced the peptides using a mass spectrometer equipped with an electrospray
ion source. The proteins from these spots were mainly involved in basic
metabolism like glycolysis (enolase and triosephosphate isomerase),
gluconeogenesis (phosphoenolpyruvate carboxykinase), citric acid
cycle (cytosolic malate dehydrogenase), electron transport-chain
(mitochondrial malate dehydrogenase), and
Some of the more obvious increases in spot size and density could be seen
in the lower right corner of the 15% (w/v) gels. Here, in the region of pI
from 8 to 10 and molecular mass of around 10 kD, several spots appeared
clearly first after 4 d of germination, and they also increased to d 6.
Analysis of four spots from this region
(Fig. 3) resulted in the
discovery of three different LTPs, two of them being the previously
characterized ElLTP1 and ElLTP2 (Edqvist
and Farbos, 2002
Previously, we found ElLTP1 and ElLTP2 transcripts mainly in a fraction
from germinating seeds containing cotyledons and endosperm
(Edqvist and Farbos, 2002
Seedlings were collected 1.5, 2.5, 4, 6, and 7 d after sowing (Fig. 6, top panel). The endosperm was separated from the cotyledons, and proteins were extracted from both tissues. Proteins were also extracted from adult plant tissues, such as leaves, stems, and roots. The amounts of both ElLTP1 and ElLTP2 are increasing in the endosperm during seed germination. The increase is most evident for ElLTP2 (Fig. 6, bottom panel). In the cotyledons, ElLTP1 protein shows a pattern resembling that of the endosperm, thus the amounts are increasing from d 1.5 to d 7. In the light of the negative results from the northern blots, we were surprised to obtain a positive signal with the anti-ElLTP2 antibodies in the cotyledons. The anti-ElLTP2 signal has a peak in cotyledon abundance at d 4, with very low amounts at d 1.5 and 7. We did detect neither ElLTP1 nor ElLTP2 in the tissue samples from adult plants.
Immunohistochemistry of ElLTPs in endosperm and cotyledon was performed on seedlings from 4 d after sowing. Staining with the anti-ElLTP1 antibodies is mainly localized to the vascular tissues, more specifically the vessel elements, of the cotyledons (Fig. 7, A and C), but also to the apoplastic space in the endosperm (Fig. 7B). Furthermore, distinct anti-ElLTP1 staining is observed in the region of the endosperm that is closest to the cotyledons. This region consists mainly of collapsed cells (Fig. 7A). There is a gradient of the ElLTP2 protein seen across the endosperm (Fig. 7D), with the stronger signal detected in the inner region of the endosperm close to cotyledons. This is not the case for ElLTP1, which is equally distributed in the endosperm (Fig. 7A). An overview of the ElLTP2 gradient in the endosperm is shown in Figure 7E. The outer region of the endosperm with minimal ElLTP2 abundance is shown in Figure 7F, whereas the inner region of the endosperm with high levels of ElLTP2 protein is shown in Figure 7G. The accumulation of ElLTP2 in the collapsed cell region is shown in Figure 7H. An interesting observation is that the ElLTP2 protein seems to be present inside endosperm cells located close to the collapsed cell region (Fig. 7I). We did not detect any ElLTP2 in the vessel elements in the cotyledons (Fig. 7J).
It is known that after depletion of the storage material, the endosperm
cells undergo senescence by PCD (Schmid et
al., 1999
To obtain a positive control of the assay, we also performed TUNEL assays
and DAPI counterstaining on slides treated with DNase I
(Fig. 8, C and D). TUNEL assays
done without terminal deoxynucleotidyl transferase served as the negative
controls of the experiments (Fig. 8, E and
F). It is generally accepted that a positive TUNEL assay is an
insufficient criterion for defining PCD because TUNEL positive nuclei can be
formed by nonspecific chromosome breakage
(Collins et al., 1992
Here, we have shown that ElLTP1 and ElLTP2 are accumulating in the endosperm as the endosperm cells undergo senescence by PCD. In endosperm of 4-d-old seedlings, ElLTP2 is mainly localized to inner regions of the endosperm. The TUNEL assay and DAPI staining showed that at this time point PCD had progressed to the outer regions of the endosperm. Thus, it seems that ElLTP2 is most abundant in dying tissues. Lipids and fatty acids are major building blocks for cell membranes and for the cuticular waxes. The expanding cotyledons need a sufficient supply of such building blocks to ensure proper growth and development. Many complex lipid structures of the dead cells, such as parts of cellular and organellar membranes, are probably turned over and re-used in the growing parts of the plant. This implies that there must be an active transport of lipids from the dead endosperm cells to the growing cotyledons. The ElLTP2 gradient in the endosperm may indicate a movement of ElLTP2, and perhaps also lipids, from the endosperm to the epicuticulary cell layer where the lipids are absorbed by the growing cotyledons. Interestingly, we detected anti-ElLTP2 signals in protein extracts isolated from the cotyledons, even though we did not detect any ElLTP2 transcripts there. In this context, we suggest that ElLTP2 function as a apoplastic carrier when lipid components from the senescent cells of the endosperm are relocalized to the growing cotyledons. A reasonable hypothesis is that the lipids transferred by ElLTP2 are used in epidermal growth and development.
It has previously been shown that during PCD of plant cells, proteases are
released from dying cells and may damage and cause necrosis of neighboring
cells (Beers and Freeman, 1997
In the silver-stained two-dimensional gels, several spots were shown to
correspond to ElLTP1 and ElLTP2. Moreover, to the left of the ElLTP3 spot
there is a very weak neighboring spot (Fig.
4) that we speculate also correspond to ElLTP3. These complicated
patterns indicate that there are multiple isoforms or that posttranslational
modifications are present in the LTPs. The pattern of the tandem ElLTP2 spots
resembles that of a phosphorylation where the modified, and thus more acidic
protein have a higher Mr. The tandem mass spectrometry
(MS/MS) sequence data have not supplied any further information on the reason
for the change of pI and mass. So, to obtain a better view of possible
phosphorylation sites in the LTPs of this study, ElLTP1 and ElLTP2 sequences
were submitted to the NetPhos 2.0 prediction server at the Centre for
Biological Sequence Analysis (Blom et al.,
1999 Together, these results indicate that the work initiated here is important for the future investigations into the recycling and degradation of cell components during senescence. It will be of high interest to investigate in further detail the function of LTPs during PCD. Future research will include an analysis of the expression of LTPs during PCD in other tissues than endosperm, but we will also search for interacting partners to the ElLTPs. Also, a more detailed investigation of the role of the probable posttranslational modifications could prove productive. Further, the creation of loss-of-function mutants in Arabidopsis could illuminate the physiological role of the LTPs.
Plant Material Euphorbia lagascae Spreng. seeds were soaked with tap water for approximately 4 h and then grown in moist vermiculite in a greenhouse facility during one to 7 d. Other seeds were cultivated in soil to adult plants. Tissues were stored at –80°C, for shorter periods of time, until used. The E. lagascae seeds were a kind gift from Prof. Sten Stymne (Department of Crop Science, SLU, Alnarp, Sweden).
Total protein extracts were prepared from the endosperm of 2-, 4-, and 6-d
seedlings. After disruption of the endosperm cells, using a mortar and pestle
in liquid nitrogen, total protein was extracted by a trichloroacetic acid
(TCA) method described by Tsugita and Kamo
(1999
An appropriate amount of TCA-precipitated proteins were diluted with fresh electrofocusing buffer to obtain 20 mg of TCA precipitate in 350 µL of buffer. To these 350 µL, DTT and IPG buffer (Amersham Biosciences AB) were added to concentrations of 20 mM and 0.5%, respectively. The protein solutions were loaded on to dry polyacrylamide gel strips with immobilized pH gradients of 3 to 10 (Immobiline Dry Strip, pH 3–10 NL, 18 cm, Amersham Biosciences AB) put in IPGphor strip holders placed on an IPGphor Isoelectric Focusing System (Amersham Biosciences AB). Gels were then rehydrated for 10 min, and isoelectric focusing was done according to the following scheme: 50 V for 12 h, 500 V for 1 h, 1,000 V for 1 h, and finally 8,000 V for 36,000 volthours up to a total of 38,101 volthours. Equilibration of gel strips was performed in an SDS equilibration buffer (50 mM Tris-Cl, pH 8.8, 6 M urea, 30% [w/v] glycerol, 2% [w/v] SDS, and bromphenol blue) with DTT (10 mg mL–1) for 15 min and then with iodoacetamide (25 mg mL–1) for 15 min. Second dimension gel electrophoresis was performed on 15% and 12.5% (w/v) polyacrylamide gels (0.4 M Tris, pH 8.8, and 1% [w/v] SDS) with a low Mr protein standard (SDS-PAGE Molecular Weight Standards, Bio-Rad Laboratories, Hercules, CA) used according to manufacturer's instructions, using a 0.1% (w/v) SDS running buffer (25 mM Tris, 192 mM Gly, and 0.1% [w/v]SDS).
Gels were stained with silver nitrate essentially according to a protocol
by Shevchenko et al. (1996
A slightly modified version of a method described by Wilm et al.
(1996
Peptide pellets were resuspended in 1 µL of 80% (v/v) formic acid and
then quickly diluted to an 8% (v/v) solution by adding 10 µL of water.
Peptide solutions were sonicated for 5 min before loading 5 µL of it to a
nanoelectrospray glass capillary (Protana Engineering A/S, Odense, Denmark)
with an R2 resin (POROS 20 R2, Applied Biosystems, Foster City, CA), binding
proteins by hydrophobic interactions. R2 capillaries were prepared by loading
1.5 µL of R2 suspension, giving approximately 300 to 400 nL of resin, to
the capillary. Excess fluid was removed by centrifugation. The resin was
washed twice with 5 µL of 0.5% (v/v) formic acid. After the wash solution
had been centrifuged through the capillary the peptide sample was added
followed by two washes with 5 µL of 0.5% (v/v) formic acid. Peptides were
eluted, by adding 0.5 µL of 25% (v/v) acetonitrile in 0.5% (w/v) formic
acid and then 0.5 µL of 50% (v/v) acetonitrile in 0.5% (w/v) formic acid
followed by centrifugation, into Au/Pd-coated nanoelectrospray glass
capillaries (Protana Engineering A/S). This method is a modified version of
that described by Wilm et al.
(1996
Peptide sequences obtained by MassLynx were subjected to BLAST using blastp
(Altschul et al., 1997
E. lagascae total RNA was isolated from the endosperm and
cotyledons of 4-d-old seedlings and from young flowers and seed pods from
adult plants. Isolation was performed using a guanidine hydrochloride method
previously described by Logemann et al. (1987). For northern-blot analysis, 10
µg of total RNA from each sample was dissolved in sample buffer (20
mM MOPS, 1 mM EDTA, 5 mM sodium acetate, 50%
[v/v] formamide, and 2.2 M formaldehyde, pH 7.9) and separated by
electrophoresis on an agarose gel (1.2% [w/v] agarose, 3% [v/v] formaldehyde,
20 mM MOPS, 1 mM EDTA, and 5 mM sodium
acetate). The RNA was transferred onto a nylon membrane (HybondN+, Amersham
Biosciences) and fixed to the membrane by UV-cross-linking (UVC 500, Hoefer)
according to the manufacturer's instructions. Immobilized RNA was hybridized
with gene-specific probes from the ElLTP1 and ElLTP2 genes
(Edqvist and Farbos, 2002
On the basis of the three-dimensional structures of maize (Zea
mays) and rice (Oryza sativa) LTPs
(Lee et al., 1998
Total protein extracts were prepared from endosperm and cotyledon from
seedlings collected 1.5, 2.5, 4, 6, and 7 d after sowing. Additionally,
protein extracts from stems, leaves, and roots were generated from adult
plants. Ten milligrams of each tissue sample was grinded in 100 µL of
2x SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4% [v/v] SDS,
20% [v/v] glycerol, 0.01% [w/v] bromphenol blue, and 20 mM DTT) and
then diluted with 100 µL of phosphate-buffered saline (PBS; 80
mM disodium hydrogen phosphate, 20 mM sodium dihydrogen
phosphate, and 100 mM sodium chloride) at pH 7.4. Before loading on
a gel, the sample was heated to 90°C for 5 min and then centrifuged. Five
microliters, corresponding to 250 µg of fresh tissues, from each sample was
loaded to the gel as well as a protein standard (MultiMark Multi-Colored
Standard, Invitrogen), used according to manufacturer's instructions. Proteins
were separated by electrophoresis on 8% to 16% (w/v) SDS-polyacrylamide gels
(Novex Pre-Cast Gels, Invitrogen; Laemmli,
1970
Four-day-old seedlings were used for all immunohistochemistry experiments. The root and hypocotyl were removed from the seedling with a knife. After that the endosperm with comprised cotyledons were fixed (4% [w/v] paraformaldehyde and 0.25% [w/v] glutaraldehyde in 0.1 M phosphate buffer pH 7.0) for approximately 16 h in room temperature. Subsequently, the seedlings were rinsed in water and then dehydrated in a graded ethanol series from 70% to 99.5%. Ethanol was finally completely removed by soaking the tissue in two changes of xylene for 1 h each before imbedding in Paraplast Plus (Sigma-Aldrich, St. Louis). Sections of 5 µm were cut using a steel blade (Rotary Microtome HM330, Microme, Heidelberg Germany), transferred to a 37°C water bath to smooth down, and then put on 3-aminopropyltriethoxysilane (Sigma-Aldrich)-precoated glass slides. Slides were incubated overnight at 37°C. Sections were deparafinized by washing in xylen for 20 min and then rehydrated in an ethanol series followed by washing in water for 1 min. For immunohistochemistry, sections were rinsed in PBS at pH 7.4 for 5 min followed by blocking of unspecific binding using PBS with 5% (w/v) dry-milk powder. Subsequent to blocking, primary antibodies from affinity-purified rabbit sera, diluted in PBS with 5% (w/v) dry-milk powder, were distributed to the sections. Detection of primary antibodies was performed using the LSAB detection kit from DAKO (Glostrup, Denmark), according to the manufacturer's instructions. Counterstaining was performed using Mayers hematoxylin (Histolab, Göteborg, Sweden), coloring all nuclei blue, according to instructions. All washes were in PBS at pH 7.4.
DNA fragmentation was detected in 5-µm sections of the same material
used for immunocytochemistry, by TUNEL analysis
(Gorczyca et al., 1993
We gratefully acknowledge the technical support of Ingrid Schenning, Ulla Pihlgren, and Ingrid Eriksson. Dr. Håkan Larsson is recognized for consultation concerning protein extraction and peptide sequence determination. Dr. Lada Filonova and Dr. Peter Bozhkov are acknowledged for helping us with the TUNEL assays. We thank Dr. Kristina Blomqvist for valuable comments on the manuscript. Received January 27, 2003; returned for revision February 28, 2003; accepted March 10, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.020875.
1 This work was supported by the Carl Trygger Foundation, by the Magnus
Bergvall Foundation, and by the AgriFunGen Research Program in Functional
Genomics at Swedish University of Agricultural Sciences. * Corresponding author; e-mail Johan.Edqvist{at}vbsg.slu.se; fax 46–18–673279.
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TOP
ABSTRACT
RESULTS
-helices tightly
connected by eight Cys residues forming four sulfide bridges. The four helices
form a hydrophobic cavity, which is the binding site for fatty acids,
acyl-CoA, or phospholipids (Lee et al.,
1998
-oxidation (glyoxysomal
