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Low Temperature Stress Modulated Secretome Analysis and
Purification of Antifreeze Protein from Hippophae rhamnoides,a
Himalayan Wonder Plant
Ravi Gupta and Renu Deswal*
Molecular Plant Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, Delhi-110007, India
*
SSupporting Information
ABSTRACT: Plants' distribution and productivity are adversely
affected by low temperature (LT) stress. LT induced proteins
were analyzed by 2-DE-nano-LC−MS/MS in shoot secretome of
Hippophae rhamnoides (seabuckthorn), a Himalayan wonder shrub.
Seedlings were subjected to direct freezing stress (−5°C), cold
acclimation (CA), and subzero acclimation (SZA), and extracellular
proteins (ECPs) were isolated using vacuum infiltration. Approx-
imately 245 spots were reproducibly detected in 2-DE gels of LT
treated secretome, out of which 61 were LT responsive. Functional
categorization of 34 upregulated proteins showed 47% signaling,
redox regulated, and defense associated proteins. LT induced
secretome contained thaumatin like protein and Chitinase as putative antifreeze proteins (AFPs). Phase contrast microscopy with
a nanoliter osmometer showed hexagonal ice crystals with 0.13 °C thermal hysteresis (TH), and splat assay showed 1.5-fold ice
recrystallization inhibition (IRI), confirming antifreeze activity in LT induced secretome. A 41 kDa polygalacturonase inhibitor
protein (PGIP), purified by ice adsorption chromatography (IAC), showed hexagonal ice crystals, a TH of 0.19 °C, and 9-fold
IRI activity. Deglycosylated PGIP retained its AFP activity, suggesting that glycosylation is not required for AFP activity. This is
the first report of LT modulated secretome analysis and purification of AFPs from seabuckthorn. Overall, these findings provide
an insight in probable LT induced signaling in the secretome.
KEYWORDS: seabuckthorn, secretome, cold stress, 2-DE, nano-LC−MS/MS, extracellular proteins, antifreeze proteins
■INTRODUCTION
Low temperature (LT) stress decreases the productivity and
restricts the distribution of crops. LT stress can be broadly
categorized into cold stress and freezing stress.
1
Plant’s
responses to freezing stress are entirely different: some can
tolerate extracellular ice formation and are referred as freezing
tolerant, while others prevent freezing by supercooling their sap
and come under the category of freeze avoiding.
2
Freezing
tolerant plants acquire tolerance to freezing temperatures
during cold acclimation (CA), which is the period when they
are exposed to low but nonfreezing temperatures. Moreover,
freezing tolerance of the plants can be further enhanced by
exposure to moderate subzero temperatures following cold
acclimation, a process known as subzero acclimation (SZA) or
second phase hardening.
3
CA in plants has been extensively
studied, but reports about the SZA are limited.
3
Furthermore,
most of the studies on CA have focused on gene expression
analysis, which has a relatively limited scope in functional
genomics.
4
Proteins are the real executors as well as final
reflectors of the gene expression. Therefore, it is appropriate to
know the proteome rather than the genome. Moreover, sub-
proteome analysis could be very beneficial in resolving the low
abundance targets by reducing the complexity of total pro-
teome. Cold modulated subproteomes of chloroplast, mitochondria,
nucleus, and plasma membrane have already been analyzed.
5−9
These studies showed that cold stress negatively affects vital
processes like photosynthesis and respiration as suggested by
degradation of stromal and key matrix enzymes of chloroplast
5
and mitochondria,
6
respectively. Analysis of nuclear proteome
showed that out of 184 identified proteins, 54 were cold
responsive.
7
Plasma membrane has sensors to sense the
temperature and then respond by changing the membrane
fluidity. The cold induced plasma membrane targets were mainly
responsible for membrane repair, membrane protection, and
proteolysis.
8
However, more subproteomes/organelles need to
be analyzed to know the complete LT responsive/regulatory
proteins repertoire and the signaling mechanisms.
Apoplast is a dynamic and complex compartment of the cell
where ice is formed at subzero temperatures to prevent lethal
cell damage. It contains the ice interacting proteins that are
involved in inhibiting or stimulating the ice crystal growth.
Besides, these extracellular proteins (ECPs) could be involved
in signal perception, cell to cell communication, and signal-
ing to provide defense against both biotic and abiotic stress.
10
Unfortunately, the molecular mechanisms and components of
Received: September 19, 2011
Published: April 10, 2012
Article
pubs.acs.org/jpr
© 2012 American Chemical Society 2684 dx.doi.org/10.1021/pr200944z |J. Proteome Res. 2012, 11, 2684−2696
the signaling in the apoplast during LT stress are poorly
understood.
Overwintering plants secrete antifreeze proteins (AFPs) in
the apoplast to keep a check on the growth of the ice crystals.
11
AFPs bind to the ice crystals and prevent accretion of water
molecules to the growing crystal planes in a noncolligative
manner. Thermal hysteresis (TH) and ice recrystallization
inhibition (IRI) are the two independent properties of AFPs, by
virtue of which they control the ice crystal size.
12
AFPs depress
the nonequilibrium freezing temperature of water below
the melting temperature, known as thermal hysteresis (TH).
TH of plant AFPs is comparatively lower (0.1−0.5 °C) than
fish (2 °C) and insect (2−5°C) AFPs.
13
It seems that the main
function of these proteins is to inhibit ice crystal growth rather
than preventing the ice formation in plants. Ice crystals grow in
size because of ice recrystallization (smaller ice crystals join to
form bigger ones). AFPs irreversibly bind to the ice crystals and
modify their shapes and growth. Ice crystals are hexagonal in
the presence of AFPs, while they are disk shaped in their
absence. AFPs increase survival of plants by inhibiting the ice
crystal growth, slowing down the ice propagation in tissue,
13
and cryoprotecting the enzymes necessary for metabolism.
14
Some of the plant AFPs are homologous to pathogenesis-
related (PR) proteins.
15
Cold induced PR-proteins (chitinases
and glucanases) retained their partial hydrolytic activity in
addition to the AFP activity.
15,16
Most of the AFPs have low
molecular weight and are apoplastic except AFP from Solanum
dulcamara, which is 67 kDa and is cytoplasmic.
17
Interestingly,
most of the AFPs are purified from the herbaceous plants;
reports about the woody plant AFPs are few.
14
Hippophae rhamnoides (seabuckthorn) is a deciduous shrub
of Himalayas. It is considered as a “wonder”plant because of its
multiple uses in biomedicine, nutraceuticals, cosmetics, and
food industries.
18
Besides a plethora of medicinal properties,
it can also withstand multiple abiotic stress conditions like
salinity, drought, UV−B radiation, and cold. Seabuckthorn has
a capability to survive at −40 °C and thrives well under cold
desert conditions. Therefore, it could be a good system for
studying its LT tolerance, and the valuable information gener-
ated can be used to develop cold/freezing stress tolerant trans-
genic crops. Such crops could be introduced in snow-clad land
masses, which could then be brought under cultivation.
Proteomics and genomics analyses of seabuckthorn for under-
standing its abiotic stress tolerance traits are still unexplored
areas. There are very few reports dissecting the molecular
mechanism of its cold hardiness.
19,20
A major bottleneck in
using seabuckthorn as a research material is the germination of
seeds in the laboratory conditions. It takes several months and
needs special presoaking treatments.
21,22
A rapid and simple
seed germination procedure is described in the present report.
Relatively fewer studies are reported for secretome analysis in
planta because of difficulties in the extraction of pure ECPs.
10
Here we describe an extraction procedure for isolating con-
tamination free ECPs. MS identification of LT induced ECPs
resolved on 2-DE gels showed the presence of putative AFPs
(PR proteins that could have antifreeze activity). Interestingly,
the antifreeze activity has not been reported in seabuckthorn to
date. To confirm the presence of AFPs, antifreeze activity was
tested in the ECPs, and to validate, these were purified using
the ice adsorption chromatography (IAC).
The goal of this study was to dissect the components and the
probable mechanisms of LT stress signaling in the secretome.
■EXPERIMENTAL SECTION
Plant Growth Conditions
H. rhamnoides berries were harvested from Keylong, Spiti Valley
of Himachal Pradesh, India. Seeds were removed from the
berries, washed, dried, and stored in a desiccator at RT. For
germination, seeds were washed with 1% teepol and surface
sterilized with 70% ethanol for 5 min. These were soaked for
1−5 days in the deionized water. After incubating for one day
in the dark, these were plated in wet germination paper rolls
and transferred to a B.O.D. incubator at 24 ±2°C under white
fluorescent light (270 μmol/m2/s, 16 h light/8 h dark).
Seedlings were watered regularly and screened for any infection
from time to time. Increasing the seed soaking duration from 1
to 5 days showed a linear increase in the germination. Maximum
(62%) germination was observed in 5 days of soaked seeds, while
it decreased by 10% after 6 days soaking (Figure S1A, Supporting
Information (SI)). Growth of seedlings was best at 20 days. One
such roll is shown in Figure S1B (SI). The germination paper
was changed twice a week to check for accumulation of inhibitors
(alkaloids and phenolics) released by seeds and to avoid any
infection (Figure S1C (SI)).
Cold Acclimation and Subzero Acclimation
For CA, 20 day old seedlings were kept at 4 °C in same light
conditions for 1 day and 5 days. SZA was given by transferring
cold acclimated seedlings to −5°C under dim light (35 μmol/
m2/s, 8 h light/16 h dark) for 24 h. One set of seedlings were
given direct −5°C for 24 h in nonacclimated conditions. For
recovery, direct −5°C treatment was followed by incubation at
24 °C for 24 h.
Freezing Survival Test and Relative Electrolyte Leakage
Test for Detecting Freezing Tolerance
Seabuckthorn seedlings were subjected to the freezing survival
test under nonacclimated, cold acclimated, and subzero accli-
mated conditions. LT treatments were given in three sets of 10
seedlings each. Survival rates were calculated after allowing the
seedlings to recover at 24 °C for 3 days.
For REL assay, seedlings were kept in small vials immersed
in 10 mL of deionized water. LT treatments were given as
described earlier. The initial conductivity (Ci) of the water was
measured at room temperature (RT) and the final conductivity
(Cf) was measured after boiling for 10 min. REL was calcu-
lated as the percentage of conductivity before and after boiling
(Ci/Cf×100) using a conductivity meter (Mettler Toledo).
Three biological replicates were performed for both survival
test and REL measurements. Statistical analysis was performed
by one-way ANOVA and post hoc Tukey HSD test, applied for
multiple paired comparisons. A value of p< 0.05 was con-
sidered statistically significant.
Secretome Extraction
The ECPs were isolated using vacuum infiltration method.
11
Shoots of control and stressed seedlings were cut into 1.0−
1.5 cm pieces. These were rinsed several times with deionized
water to remove the cytoplasmic proteins. After washing, these
sections were vacuum infiltrated at 10 mmHg for 30 min with
different buffers (deionized water, 50 mM Tris pH 7.4, 20 mM
ascorbic acid with 20 mM CaCl2, 20 mM ascorbic acid with
20 mM MgCl2, and 20 mM ascorbic acid). These were dried
and placed in a syringe barrel in a falcon tube. The ECPs were
collected by centrifugation at different speeds (2000−6000g)
for 10 min. Proteins were acetone precipitated and quantified
using Bradford’s method.
23
For total protein extraction, tissue
Journal of Proteome Research Article
dx.doi.org/10.1021/pr200944z |J. Proteome Res. 2012, 11, 2684−26962685
was extracted in 1:1 (wt/vol) buffer (50 mM Tris pH 7.4,
20 mM EDTA, 30% glycerol, and 5 mM PMSF). Extract was
centrifuged at 12 500 rpm at 4 °C for 25 min (Beckman
Coulter, Allegra 64R). Supernatant was acetone precipitated
after filtering through two layers of muslin cloth. The pellet was
dissolved in the glycine buffer pH 8 and used for the glucose-6-
phosphate dehydrogenase (G6PDH) activity assay.
Testing ECPs for Cytoplasmic Contamination
G6PDH activity assay was performed to test cytoplasmic
contamination in ECPs of control and stress treated seedlings.
For G6PDH assay, 40 μL of protein extract was added in 1 mL
of the reaction mixture containing 55 mM Tris pH 7.8, 3.3 mM
MgCl2, 6 mM NADP, and 0.1 M of glucose-6-phosphate.
Absorbance was taken at 340 nm for 5 min at 25 °C using
UV−visible spectrophotometer (DU 730, Beckman Coulter),
and enzyme activity was calculated as described by Noltmann
et al.
24
G6PDH assay was performed in ECPs and crude extract.
Percentage cytoplasmic contamination was calculated as the
activity in the apoplastic extract over the activity in crude
extract. Purified G6PDH from Leuconostoc mesenteroides was
used as a positive control.
Construction of 2-DE Reference Map of LT Induced ECPs
ECPs were resolved on SDS-PAGE following Laemmli.
25
A“cleanup”step was included to remove contaminants. One
volume of protein extract was mixed with four volumes of
methanol followed by addition of one volume of chloroform.
Three volumes of deionized water were added, and the mix was
centrifuged at 12 000 rpm (1−15k, Sigma) for 5 min at RT.
Supernatant was carefully discarded, keeping the interphase
protein layer. To the protein disk, three volumes of methanol
were added, and it was again centrifuged at 12 000 rpm for
5 min. After removing the supernatant, the pellet was dried in a
vacuum chamber (Millipore). Each step was followed by vigorous
vortexing to mix the contents.
Protein pellets after “cleanup”step were mixed with 1X
sample buffer, boiled for 3 min, and resolved on a 15% SDS-
PAGE gel (Hoefer MiniVE). Electrophoresis was carried out at
120 V for about 2 h, and the gels were silver stained. For 2-DE,
250 μL of rehydration buffer containing 250 μg of protein was
loaded onto 13 cm IPG strips (pH 3 to 10, nonlinear gradient,
GE Healthcare) by rehydration loading overnight at RT. IEF
was performed on an EttanIPGphore isoelectric focusing sys-
tem (GE Healthcare) for 27.5 kVh. After IEF, the strips were
reduced in an equilibration buffer (6 M urea, 50 mM Tris pH
8.8, 30% glycerol, 2% SDS, and 0.002% BPB) containing 1%
DTT as the first step and then alkylated by 2.5% iodoacetamide
as the second step. For the second dimension, proteins
were resolved on 15% SDS-PAGE using Hoefer SE 600 Ruby
(GE Healthcare). The gels were silver stained.
26
Five biological
replicates were performed for freeze treated samples, and 2-DE
gels of other samples were performed with three biological
replicates.
Image Acquisition and Data Analysis
Gels were scanned using Alpha Imager (Alpha Innotech
Corporation). ImageMaster2DPlatinum software (ver 6.0; GE
Healthcare, Sweden) was used to analyze the silver stained
2-DE gels. A first level matchset was created in which master
gels were developed from five replica gels of freeze treated
samples and three replica gels of other samples that have
correlation coefficient of at least 0.8. Volume of each spot was
normalized in percentage spot volume mode to compensate for
the differences in protein loading and gel staining. After spot
detection in these gels, a second level of matchset was created
in which master gels of different treatments were matched and
compared. The intensity of a given protein spot was expressed
in terms of its volume, which was defined as the sum of the
intensities of all pixels constituting the spot in the image.
Student’st-test was performed to observe the significant changes
(p< 0.05) between the intensities of the spots.
Protein Identification Using Nano-LC−MS/MS
Protein identification was carried out at The Centre for Genomic
Applications (TCGA), New Delhi, India using Agilent 1100
series 2D nano-LC−MS/MS. LT induced polypeptides were
excised from the gel and destained. In gel reduction was carried
out using 10 mM DTT in 100 mM ammonium bicarbonate at
56 °C for 30 min. Alkylation of reduced proteins was done using
50 mM iodoacetamide in 100 mM ammonium bicarbonate for
30 min in the dark. Gel pieces were washed with 1:1 ammonium
bicarbonate and acetonitrile (ACN) solution and dehydrated
using 100% ACN for 5 min. Gel pieces were digested with 5 μL
of trypsin solution (20 ng/μL, gold mass spectroscopy grade,
Promega, Madison, USA) in 50 mM ammonium bicarbonate pH
7.8 for 16 h at 37 °C. Tryptic digested peptides were extracted
twice with 0.1% trifluoroacetic acid (TFA) and were separated
by HPLC using Agilent 1100 NanoLC-1100 system (Agilent,
Palo Alto, CA, USA) combined with a microwell-plate sampler
and thermostatted column compartment for preconcentration
(LC Packings, Agilent). Samples (6 μL) were loaded on the
Zorbax 300SB-C18 column (150 mm ×75 μm, 3.5 μm) using a
preconcentration step in a micro-precolumn cartridge (Zorbax
300SB-C18, 5 mm ×300 μm, 5 μm) at a flow rate of 5 μL/min.
The precolumn was connected with the separating column, and
after 5 min, a multistep gradient (3% until 5 min, 15% for 5−
8 min, 45% for 8−50 min, 90% for 50−55 min, 90% for 55−
70 min, then again 3% for 71 min) was started. Formic acid
(0.1%) in water and in 90% ACN were used as buffers. An LC−
MSD Trap XCT with a nanoelectrospary interface (Agilent)
operated in the positive ion mode was used for MS. Ionization
(1.5 kV ionization potential) was performed with a liquid
junction and a noncoated capillary probe (New Objective,
Cambridge, USA). Standard Agilent tune mix was used to
calibrate the instrument. Peptide ions were analyzed by the
data-dependent method. The scan sequence consists of 1 full
MS scan followed by 4 MS/MS scans of the most abundant
ions. Data was analyzed using Agilent ion trap analysis software
(ver 5.2). The peak lists were submitted to MASCOT (ver 2.1)
search engine (http:/www.matrixsciences.com) and searched
against the NCBInr database. The search parameters were as
follows: mass values, monoisotopic; protein mass, unrestricted;
fixed modifications, carbamidomethylation; variable modifica-
tion, methionine oxidation; peptide mass tolerance, ±1.2 Da;
fragment mass tolerance, ±0.6 Da; maximum trypsin missed
cleavage, 1; and instrument type, ESI-TRAP. Only significant
hits, as identified by the MASCOT probability analysis (p< 0.05)
were accepted.
Enzyme Assays
Glyoxylase 1 assay was performed according to Deswal and
Sopory with slight modifications.
27
In brief, 20 μL of the pro-
tein extract was added in 1 mL reaction mixture containing
200 mM sodium phosphate buffer pH 7.6, 3.5 mM methyl-
glyoxal, 1.6 mM glutathione, and 5 mM NiCl2(instead of
16 mM MgSO4). Absorbance was taken at 240 nm for 5 min.
One enzyme unit is the amount of enzyme catalyzing the
Journal of Proteome Research Article
dx.doi.org/10.1021/pr200944z |J. Proteome Res. 2012, 11, 2684−26962686
formation of micromole of S-lactoyl glutathione per minute per
milligram. Chitinase assay was performed using chitin azure as a
substrate.
28
Briefly, 50 μL of the sample was added in 950 μLof
200 mM sodium phosphate buffer pH 7 containing 10 μg of the
substrate. Reaction mixture was incubated at 37 °C for 24 h
with constant shaking. Absorbance was taken at 570 nm after
centrifuging it at 16000gfor 10 min. One unit of Chitinase is
the change in absorbance of 1.0 in 24 h at 570 nm. For SOD
assay, 50 μL of protein was added in 1 mL of reaction mixture
containing 100 mM triethanolamine (pH 7.4), 100 mM EDTA,
50 mM MnCl2, 7.5 mM NADH, and 10 mM mercaptoethanol.
Decrease in absorbance was recorded at 340 nm for 15 min.
One unit of enzyme is the amount of SOD capable of inhibiting
50% rate of NADH oxidation observed in the control.
29
Three
biological and technical replicates were performed for each
assay.
Antifreeze Activity Assay
IRI and TH activities were analyzed using sucrose sandwich
splat assay
30
and nanolitre osmometer.
11
All antifreeze activities
were carried out at a protein concentration of 0.2 mg/mL. For
IRI assays, proteins were solubilized in 30% sucrose in 20 mM
ammonium bicarbonate pH 8 and were sandwiched between two
round coverslips. These coverslips were snap frozen at −80 °C
and then transferred to a glass viewing chamber maintained at
−6°C. Ice crystals were allowed to anneal for 2 h and then
photographed using a Nikon eclipse 80i microscope with a DS-
Fi1 camera (Nikon) and NIS-elements F-package (ver 3.0)
software. For the quantification of the IRI activity, the dia-
meters of the ice crystals in each image were measured using
Image J software. TH and the shapes of ice crystals were
analyzed using nanolitre osmometer. In brief, nanolitre volumes
of the samples were loaded into the wells of the sample holder
disk and kept on a freezing stage mounted on the stage of a
phase-contrast microscope. Temperature was controlled by a
nanoliter osmometer (Otago Osmometers, Dunedin, New
Zealand). The samples were flash frozen at −20 °C to form a
population of small ice crystals and then thawed until only a
single ice crystal remained in the well. Temperature was
gradually decreased, and the morphology of the ice crystals was
photographed with the 20×objective. The temperature at which
this ice crystal grew and shrunk was taken as the freezing and
melting point of the sample, respectively. TH was calculated as the
difference between the melting and freezing temperatures.
ECPs were treated with Proteinase-K (Sigma, 1 mg/mL)
overnight at 20 °C. For heat treatment, ECPs were incubated in
boiling water for 10 min and then centrifuged to settle the
precipitate. Five biological replicates were performed for all
antifreeze assays with each assay performed in triplicates.
Purification of Antifreeze Proteins
IAC was done for purification of antifreeze polypeptide.
31
ECPs
from −5°C treated seedlings were isolated and acetone preci-
pitated. Pellet was dissolved in 20 mM ammonium bicarbonate
pH 8. The cold/brass finger was seeded with a thin layer
of ice, and it was immersed in the prechilled protein extract
(≈50 mL). Temperature of the coldfinger was gradually de-
creased using a refrigerated water bath (GP150, Grant Instru-
ments, Cambridge, U.K.) from −3°Cto−7°C over 30 h with
the help of LabWise software. Protein extract was gently mixed
on a magnetic stirrer. After completion of the IAC, ice fraction
was removed from the coldfinger by increasing the temperature
to 1 °C. The ice fraction was melted, and the proteins were
lyophillized (Labconco). For identification of antifreeze
proteins, ice binding proteins were eluted in 50 mM Tris pH
7.6 from the 12% preparative native gels.
11
Treatment with Glycosidase
For deglycosylation, 4 μg of purified PGIP was treated with
1 unit of N-terminal glycosidase (peptide-N-glycosidase,
PNGase-F, Sigma) and incubated at 37 °C overnight following
manufacturer’s instructions.
■RESULTS AND DISCUSSION
Analyzing the Effect of LT Stress on Seabuckthorn
Seedlings
Effect of freezing stress on nonacclimated, cold acclimated, and
subzero acclimated seabuckthorn seedlings was observed by
freezing survival test and REL measurement. Survival rates of
control, 1 day, and 5 days cold acclimated seedlings were 100%.
However, when seedlings were exposed to freezing stress in
nonacclimated condition, survival rate was 81.65%. This sur-
vival rate of direct −5°C treated seedlings was much higher
than LT50, suggesting that seabuckthorn is able to tolerate
direct freezing temperatures as well. Droop test analysis also
supported this observation, as seedlings were able to tolerate
freezing temperatures in nonacclimated conditions up to 5 days
without any visible sign of drooping. One-way ANOVA
followed by Tukey post hoc test showed insignificant difference
(p> 0.05) in survival rates between the seedlings that were
directly subjected to freezing stress and the seedlings that were
exposed to freezing stress after 1 day of CA. However, when 5
days cold acclimated seedlings were subjected to freezing stress,
survival rates were increased significantly to 94.5% (Figure 1A).
These results showed that although seabuckthorn is able to
tolerate direct freezing temperatures in nonacclimated condi-
tions, its freezing tolerance can be enhanced further by pro-
longed cold acclimation. Consistent with the results of survival
test, REL measurement showed a similar trend, as freezing
injury was significantly less after 1 and 5 days of CA (34.29 and
26.86%, respectively) in comparison with the nonacclimated
seedlings exposed to freezing stress (35.15%, Figure 1B).
Extraction of Contamination Free Secretome
Analyzing secretome to understand the mechanism of freezing
tolerance is pertinent, as this is the communication channel of
the cell with the environment. Also, this is the region of the cell
that is most affected by freezing stress due to formation of ice
crystals. For secretome analysis, the first and the most crucial
step is the extraction of contamination free ECPs.
Classical procedures of ECPs extraction involve vacuum
infiltration followed by centrifugation (800−2000g).
10
How-
ever, this method yields low concentration of ECPs. We used
combinations of buffers and differential centrifugal speed to
improve the yield of ECPs. Out of different buffers used for
extraction (as mentioned in experimental section), a com-
bination of ascorbic acid and calcium chloride gave best results,
as Rubisco (a chloroplastic protein) was absent and the protein
yield was 1.74-fold higher than ascorbic acid alone at 2000g
(Figure 2A, lane 3). Extraction in Tris and deionized water
showed Rubisco contamination (Figure 2A, lane 5 and 6). For
increasing the yield further without compromising on the
purity, higher centrifugal force was applied (Figure 2B) with
ascorbic acid and calcium chloride. Yield increased (1.9-fold) at
4000g, but it decreased at 6000g(Figure 2B, lane 6). At 5000gand
6000g, a slight greenish extract was obtained, indicating membrane
disruption and pigment leakage. Therefore, 4000gwas selected for
Journal of Proteome Research Article
dx.doi.org/10.1021/pr200944z |J. Proteome Res. 2012, 11, 2684−26962687
the secretome extraction. For the rest of the experiments, ECPs
were extracted in ascorbic acid and calcium chloride at 4000g.
Purity Assessment of the Secretome
High centrifugal force (4000g)couldcausemembrane
disruption leading to cytoplasmic contamination. Therefore, it
was crucial to test the purity of the ECPs. G6PDH assay was
conducted to estimate the percent cytoplasmic contamina-
tion in ECPs of control, nonacclimated, cold acclimated, and
subzero acclimated seedlings. Control ECPs did not show
G6PDH activity. However, ECPs obtained from all the LT
treated seedlings showed G6PDH activity that was restricted to
less than 3% of the total G6PDH activity in the crude, which is
within the permissible limit (Figure 2C).
32
According to Song
et al., up to 10% cytoplasmic contamination is acceptable for
secretome analysis.
33
LT Stress Differentially Modulated the Shoot Secretome
In order to determine the changes in secretome during CA,
SZA, and direct freezing in nonacclimated conditions, ECPs
were isolated using a modified vacuum infiltration protocol and
were resolved on the 2-DE gels. Image analysis of 2-DE gels of
ECPs from the control, CA (1d), direct −5°C, CA (1d)
followed by −5°C, CA (5d), CA (5d) followed by −5°C, and
recovered seedlings apoplastic extracts reproducibly showed
243, 249, 255, 263, 238, 251, and 227 spots, respectively. The
quantitative image analysis in combination with statistical tests
showed that a total of 61 (25%) spots changed in abundance by
more than 2-fold (p< 0.05) in at least one LT treatment
(Figure 3). Out of these 61 differentially expressed spots, 26
spots showed higher abundance, 13 spots showed decreased
abundance, and the remaining 17 spots showed a mixed pattern
of expression during LT stress treatment. Six gel regions show-
ing differential abundance of spots after LT were magnified to get
a clearer view of the abundance patterns (Figure 4). The
2-DE gel profile of −5°C treated seedlings showed that a majority
(63%) of the spots were confinedtotheacidicpI.AsECPsare
mainly acidic in nature, this reaffirms the quality of the secretome.
Identification and Prediction of the Secretory Nature of the
LT Induced Proteins
Spots that showed more than 2-fold increased abundance,
student’st-test (p< 0.05), after LT stress were identified. A
total of 31 spots showing 34 proteins were identified by nano-
LC−MS/MS (Table 1). Identified proteins with only signifi-
cant ion scores (p< 0.05) and E-values (E< 0.05) were
accepted. However, the difference in theoretical and experi-
mental molecular weights and pIs of some of the identified pro-
teins may be due to lack of protein database for seabuckthorn,
protein degradation, protein isoforms, post-translational modi-
fications, and alternative splicing.
Extracellular localization, predicted using SignalP and
SecretomeP software, showed 75.75% of the identified
proteins to be extracellular, while 24.25% were nonsecretory. Out
of the 75.75% secretory proteins, 24.25% were classical secretory,
i.e., were carrying signal peptide, while the remaining 51.5% were
targeted via the nonclassical pathway(s). As nonsecretory proteins
are not the resident of apoplast, these might be imported in
response to any stimulus like stress condition(s).
Functional Classification and Clustering of the LT Induced
Proteins
On the basis of the biological roles, the identified proteins were
classified into 6 categories: redox regulation, stress tolerance,
Figure 2. Extracting contamination free extracellular proteins from
shoot. (A) SDS-PAGE profile of extracellular proteins extracted in
different buffers at 2000g. (B) SDS-PAGE profile of extracellular proteins
extracted in ascorbic acid and calcium chloride at different centrifugal
forces (2000−6000g). Polypeptide labeled as LSU is large subunits of
Rubisco. (C) G6PDH assay was performed to test the purity of ECPs.
Error bars represents standard deviation from three biological replicates.
Figure 1. Effect of CA, SZA, and direct freezing on seabuckthorn
seedlings. (A) Freezing stress was given to nonacclimated and cold
acclimated (1 and 5 days) seedlings, and survival rates were calculated
after allowing them to recover at 25 °C. (B) Effect of LT on REL of
seabuckthorn seddlings. Error bars represents standard deviation from
three biological replicates. Statistical significance was determined by
the Tukey−Kramer multiple comparisons test. Values with the same
letters are not significantly different (p> 0.05).
Journal of Proteome Research Article
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signaling, metabolism, regulation, and others (Figure S2 (SI)).
Proteins that fall in the others category were either hypothetical
or proteins with unknown functions.
As the present study examines the abundance patterns of LT
responsive proteins by different LT stress conditions, it was
very crucial to differentiate the proteins induced by direct
Figure 3. 2-DE reference gels of seabuckthorn ECPs obtained from control, CA (1d), −5°C (1d), recovery, CA (1d) followed by −5°C, CA (5d),
and CA (5d) followed by −5°C. LT treated seedlings. ECPs (250 μg) were resolved on 3−10 nonlinear IPG strips for the first dimension and 15%
SDS-PAGE for the second dimension. Gels were silver stained and analyzed by ImageMaster2D Platinum software (GE Healthcare). Spots showing
a more than 2-fold change in abundance due to LT treatments (p< 0.05) are marked by the arrows.
Figure 4. Magnified sections of 2-DE gels of the seabuckthorn shoot secretome showing differential abundance of the proteins after LT stress in a
dose dependent manner. Spot numbers in the boxes refer to the spots corresponding to Figure 3.
Journal of Proteome Research Article
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freezing, CA, and SZA. For this, the identified proteins were
subjected to clustering analysis using cluster 2.1.1 program
(http://rana.lbl.gov/EisenSoftware.htm). Six clusters were
formed on the basis of the similarities in the abundance
profiles (Figure 5).
Cluster 1 contains proteins that showed a mixed abundance
pattern during LT stress. These proteins include predicted
protein (Physcomitrella patens), hypothetical protein SORBI-
DRAFT, calmodulin, and Os10g0125700. These proteins
belonged to different functional categories and thus represent
a broader modulation of metabolic and regulatory pathways.
Cluster 2 includes proteins that seem to be freezing sensitive, as
their abundances were decreased after freezing treatment either
in nonacclimated conditions or in cold acclimated conditions.
These proteins include putative blue light receptor and
unnamed protein product (Vitis vinifera). Cluster 3 represents
proteins that are induced by direct freezing. These proteins
include GTPase-activating protein, glyoxalase 1, C-3 sterol
dehydrogenase, superoxide dismutase (SOD), aldo/keto
reductase, hypothetical protein OsI_37876, thylakoid lumenal
Table 1. Identification of the Spots That Showed ≥2-Fold Induction after Freezing Stress by Nano-LC−MS/MS
functional
category spot
no.
a
protein identified accession no. protein
score
b
M
c
SC
d
th MW/pI
exp MW/
pI
e
loc
f
redox regulation 24 Lactoylglutathione lyase or Glyoxylase 1 XM_002518424.1 161 2 9 31.7/7.63 24.4/5.5 NC
15 Superoxide dismutase P23346 64 1 3 15.2/5.64 15.3/5.1 NC
6 Thioredoxin EU056813.1 62 7 5 20.6/9.63 10.4/5.2 C
35 Aldo/keto reductase AC160516.1 52 2 4 31.1/6.15 29.9/5.3 NS
36 Putative lactoylglutathione lyase Q39366 98 1 4 31.74/5.19 33.58/5.6 NC
defense/stress
related 32 Osmotin-like protein AF304007.1 83 3 7 27.5/7.41 26.7/5.4 C
32 Thaumatin-like protein P83491 64 7 43 11.42/4.4 26.7/5.4 C
12 Chitinase XP_003597296 74 2 3 33.72/5.9 13.89/5.5 C
18 Similar to pathogenesis-related protein STH-2 AB211525.1 72 3 7 17.34/5.79 17.9/5.2 NS
7 GDSL-motif lipase/hydrolase family protein NP_177281 56 3 2 41.5/9.1 11.91/5.9 NC
37 Dessication related protein AAM65140 98 1 5 34.4/8.77 29.6/7.2 C
40 Phenylalanine ammonia lyase ABD42947 73 1 1 78.2/6 31.91/5.4 NC
30 Late embryogenesis-like protein AAU29064 53 1 8 17.32/4.51 21.73/8.8 NC
signaling 1 Calmodulin 1 DQ186609.1 57 3 14 16.9/4.16 14.54/3.1 NC
55 Calcium dependent protein kinase 23 XP_002309145 50 1 2 60.14/5.91 42.3/8.1 NC
3 GTPase activating protein AAQ54568 1 18 8.68/4.96 12.1/3.5 NC
metabolism 4 C-3 sterol dehydrogenase CAL52542 70 4 1 205.2/7.22 10.9/3.2 NS
41 Sedoheptulose-1,7-bisphosphatase ACQ82818 147 6 8 42.53/5.96 36.73/5.3 NS
59 Putative blue light receptor CAC94940 52 1 1 81.9/8.67 NS
2 Putative phosphomannomutase BAD35746 54 1 3 53.7/6.99 11.46/3.2 NC
regulation 20 ATP-dependent Clp protease ATP-binding
subunit clpA homologue P84565 115 3 51 7.9/4.39 17.04/6.8 NS
11 Cysteine protease AF134152.1 70 1 11 15.6/4.12 13.54/5.3 NC
11 Translation-inhibitor protein AB082518.1 111 3 11 19.8/7.63 13.54/5.3 C
25 Pyrrolidone-carboxylate peptidase family protein NP_564721 48 1 6 24.3/5.98 26.33/5.9 NS
others 13 Thylakoid lumenal 15 kDa protein, chloroplast
(Arabidopsis thaliana)NP_566030 88 3 6 24.1/7.55 13.89/5.8 NC
48 Unknown protein (Arabidopsis thaliana) NP_191832 101 1 5 35.2/5.27 72.37/5.3 C
5 Predicted protein (Physcomitrella patens subsp.
patens) XP_001763519 56 1 2 67.3/6.78 11/3.2 NC
11 Unknown (Populus trichocarpa) ABK93605 179 6 17 21.98/8.76 13.54/5.3 NC
27 Os10g0125700 NP_001064077 49 1 1 161.8/8.15 26.1/6.3 C
57 Unnamed protein product (Vitis vinifera) CBI40282 70 1 4 43.6/4.75 42.3/8.4 NC
19 Predicted protein (Physcomitrella patens subsp.
patens) XP_001784896 73 4 3 42.8.3/5.5 17.71/5.9 NC
9 Hypothetical protein OsI_37876 (Oryza sativa
Indica Group) EEC69045 54 2 1 157.6/6.54 12.5/6.4 NS
8 Unknown protein 18 P85925 65 1 91 1.39/5.8 12.22/5.9
50 Hypothetical protein SORBIDRAFT XP_002456647 55 1 8 14.8/7.04 67.88/5.6 NC
a
Spot no. refers to the spots labeled in Figure 3.
b
Protein scores are derived from ion scores as a nonprobabilistic basis for ranking
protein hits.
c
Number of matched peptides.
d
Sequence coverage.
e
Experimental molecular weight and pIwere calculated using
ImageMaster2DPlatinum software.
f
Putative location was predicted using SecretomeP and SignalP servers. C, classical; NC, nonclassical; NS,
nonsecretory.
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15 kDa protein, and ATP-dependent Clp protease ATP-binding
subunit clpA homologue. These proteins seem to be induced by
freezing rather than the low temperature effect. Cluster 4
includes proteins that keep on increasing with increasing LT
duration. These proteins include unknown protein 18, pre-
dicted protein (Physcomitrella patens), calcium dependent
protein kinase, and thioredoxin. Proteins of cluster 5 are induced
during SZA, as abundances of these proteins are induced only
after freezing stress. These proteins include cysteine
protease, late embryogenesis-like protein, Chitinase, similar
to pathogenesis-related protein STH-2, sedoheptulose-1,7-
bisphosphatase, thaumatin-like protein, putative phospho-
mannomutase, and GDSL-motif lipase/hydrolase family
protein. Proteins of this cluster are mainly involved in stress
tolerance. Cluster 6 includes proteins that maintained a
constant abundance pattern in all the LT treatments after an
initial increase. These proteins seem to be involved in normal
LT stress tolerance response and include putative lactogluta-
thione lyase and a hypothetical protein.
Proteins Involved in the LT Stress Response in
Seabuckthorn Secretome
Stress conditions lead to an accumulation of superoxide radicals
(O2
−) from different sources in the apoplast.
34
One of the
sources is reduction of NAD+. NAD+is reduced to NADH by
different dehydrogenases (C-3 sterol dehydrogenase, spot no. 4).
This reduced NADH is oxidized to superoxide radical by NADH
oxidase. SOD (spot no. 15) converts superoxide ions to H2O2,
which is finally reduced to H2O either by catalase directly or by
ascorbate peroxidase and glutathione peroxidase. Although none
of the identified plant SOD have signal peptide, MS based
identification and significant activity modulation by freezing
observed in the present study confirmed its apoplastic
localization.
35
Role of SOD in freezing tolerance was earlier
shown in Medicago.
36
Spots no. 36 and 24 were identified as glyoxalase 1 or
lactoglutathione lyase. Interestingly, the sizes of two glyoxalase
1 vary, 24.4 and 33.58 kDa, suggesting these could be the two
different forms of glyoxalase 1 in seabuckthorn. This enzyme is
involved in detoxification of the methylglyoxal that is formed as
a byproduct of carbohydrate and amino acid metabolism and
thus is indirectly involved in the ROS metabolism. MS based
identification of the ECPs showed an increased abundance
of glyoxalase 1 after multistress response in poplar
37
and
dehydration stress in rice,
38
hinting at its apoplastic import
in stress.
In addition to the H2O2mediated signaling, two other
signaling pathways seem to be modulated in the secretome after
freezing stress. Increased abundance of the calmodulin (spot
no. 1) and calcium dependent protein kinase (spot no. 55)
indicates activation of calcium signaling pathway, while in-
creased abundance of GTPase-activating protein (spot no. 3)
showed modulation of G-protein signaling. Calmodulin is a
ubiquitous calcium sensor in plants. It binds to calcium and
stimulates downstream targets. Glyoxalase 1 is activated by
calmodulin.
39
Thus, accumulation of calmodulin by LT stress
may activate glyoxylase 1. It was earlier reported that calmodulin-1
Figure 5. Clustering analysis of LT stress modulated proteins of shoot secretome. The cluster was performed using CLUSTER 2.1.1 software
(http://rana.lbl.gov/EisenSoftware.htm) and is shown at the top. Differential abundance and functional categories of the proteins in each cluster are
depicted in lower left and right panel, respectively.
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is involved in cold stress signaling cascade in Arabidopsis.
7,40
Increased level of calmodulin-1 after cold and high wind was
also shown in Nicotiana plumbaginifolia.
41
A very encouraging observation was the presence of some
putative AFPs like thaumatin-like protein (spot no. 32) and
Chitinase (spot no. 12) in the seabuckthorn secretome.
Although the main function of these proteins is in biotic
stress, their role in cold stress is also well established.
15
These
proteins acquire antifreeze activity at the time of cold/freezing
stress and thus prevent the lethal cell damage caused by the ice
crystals. Apart from the antifreeze activity, Chitinase also retains
its partial hydrolytic activity at subzero temperatures,
16
thus
acting as a dually functioning protein.
A very high percentage of identified proteins (29%) belongs
to the “others”category; this may be due to the scarcity of pro-
tein sequences submitted for this plant in the databases.
Characterization of these could provide us interesting, uniden-
tified targets for crop manipulation.
Enzymatic Activities for the Functional Validation of the
Identified Proteins
Activities of three enzymes, glyoxalase 1, SOD (induced by
freezing), and Chitinase (induced by SZA), showing more than
20-fold accumulation by LT stress were analyzed.
Both Chitinase and SOD activities were increased during LT
stress. In SOD, there was a generalized increment in the activity
with increasing LT duration, and maximum activity (1.4-fold
higher in comparison with the control) was recorded when
freezing stress was given to 5 days cold acclimated seedlings.
Chitinase activity was maximum in freezing stress whether the
seedlings were acclimated or nonacclimated (Figure 6).
Glyoxalase 1 activity was not detected in ECPs probably
due to the presence of a different (inactive) form. Apoplastic
glyoxalase 1 may perform some different function than
cytoplasmic glyoxalase 1.
LT induced fold change in enzymatic activities were multi-
plied with the fold induction in abundance (calculated by
ImageMaster), to calculate the effective induction of these en-
zymes. Increase in the amount of the protein as well as its
activation would have a synergetic effect. Chitinase and SOD
showed 60- and 25.6-fold effective inductions after freezing
stress, justifying their high requirements.
Detection and Identification of Antifreeze Proteins
Some of the PR-proteins play very crucial roles in LT by
switching their PR-activity to antifreeze activity.
15
As putative
AFPs were identified in the LT treated secretome, to confirm
their presence, the AFP activity was analyzed by a nanolitre
osmometer coupled with a phase contrast microscope and by
sucrose sandwich splat assay. Activity assays showed freezing
stress induced antifreeze activity, which inhibits ice recrystal-
lization (average ice crystal diameter was reduced from 23.6 to
16.3 μm), forming hexagonal ice crystals with 0.13 ±0.02 °C
TH (Figure 7A,B).
ECPs when treated with proteinase-K lost antifreeze activity
(Figure 7A,B), confirming the proteinaceous nature of the
antifreeze activity. Heat treatment of ECPs obtained from
−5°C treated seedlings resulted in loss of 68% of the activity
(Figure 7A,B), suggesting that 32% of the antifreeze activity
was due to heat stable proteins. As antifreeze proteins were
detected in the freeze modulated secretome, an effort was made
to purify these.
Purification and Characterization of Antifreeze Proteins
AFPs were enriched using IAC. Out of 50 mL of ECPs in
ammonium bicarbonate, nearly 35 mL was bound to the ice
fraction after completion of IAC. Lyophilized ice bound
proteins showed a major polypeptide of 41 kDa (Figure 7C).
However, three other minor polypeptides were also observed in
the SDS-PAGE of IAC fraction. Binding of other three proteins
to the ice column was not consistent; therefore, these were not
analyzed further. Efforts are underway to enrich other AFPs by
modifying the IAC procedure.
MS identification of 41 kDa ice bound protein showed it to
be polygalacturonase inhibitor protein (PGIP). This protein
belongs to Leucine-rich-repeat (LRR) protein family, and its
main function is to protect the plant from fungal attack by
inhibiting the polygalacturonase secreted by pathogenic fungi.
Bioinformatic analysis of this protein using SignalP software
showed its targeting in the apoplast via the classical secretory
pathway. However, in our 2-DE experiments, PGIP was not
identified. This may be due to low levels of PGIP in the
secretome. To confirm the LT inducible nature of PGIP, IAC
was performed using ECPs obtained from control seedlings.
PGIP was not detected in the IAC fraction of control seedlings
Figure 6. Enzyme activities of Chitinase (upper panel) and SOD (lower panel) for functional validation of the LT induced targets (A, D). Enzymatic
activities were multiplied with the relative intensities of the spots (B, E) to calculate the effective inductions (C, F).
Journal of Proteome Research Article
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showing that PGIP binds to the column only after freezing
treatment because of its enrichment after freezing (Figure 8A).
Cold inducible nature of PGIP was earlier shown in cotton,
42
Arabidopsis,
43
Brassica napus,
44
and Chorispora bungeana.
45
Association of antifreeze activity with PGIP is well established
in carrot.
30
PGIP identified in seabuckthorn showed a high
degree (93%) of sequence similarity with carrot PGIP. PGIP
was resolved at 41 kDa in both SDS and native-PAGE,
Figure 7. Analysis of antifreeze activity in freezing stress induced seabuckthorn secretome. (A) Ice crystal morphologies, IRI assays, and (B)
quantification of these activities in buffer, BSA, control ECPs, −5°C treated ECPs, heat treated, and proteinase K treated ECPs, analyzed by phase
contrast microscope coupled with nanoliter osmometer and sucrose sandwich splat assay. (C) SDS-PAGE and (D) native gel profile showing
purification of polygalacturonase inhibitor protein by IAC. The band marked with an asterisk is PGIP. Magnification bar in nanoliter osmometry
shows 10 μm, while in splat assay it represents 50 μm. Error bars show standard deviation from five replicates.
Figure 8. (A) SDS-PAGE profile showing IAC fractions of control and −5°C treated ECPs. PGIP was not identified in IAC fraction of control
seedlings, while it is present in the IAP fraction of −5°C treated seedlings. (B) SDS-PAGE profile of PGIP showing its glycosylation and heat
stability. The band marked with an asterisk is PGIP, and the band marked with “+”showed PNGase-F. Ice crystal morphology, IRI assays (C), and
their quantification (D) in presence of PGIP, deglycosylated PGIP, heat, and Proteinase-K treated PGIP. (E) Determining IRI end point. PGIP was
serially diluted and tested for antifreeze activity. AFP activity was completely lost at a concentration of 0.012 mg/mL. Magnification bar in nanoliter
osmometry shows 10 μm, while in splat assay it represents 50 μm. Error bars show standard deviation from five replicates.
Journal of Proteome Research Article
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suggesting its monomeric nature. It was eluted from the native
gel and tested for AFP activity. Inhibition of ice recrystallization
(average ice crystal diameter was reduced from 23.6 to 2.6 μm)
and formation of hexagonal ice crystal by PGIP confirmed it to
be an antifreeze protein (Figure 8C,D). A TH of 0.19 ±0.03 °C
was observed at 0.2 mg/mL. This TH of seabuckthorn PGIP was
higher than Lolium AFPs (IAP 2, IAP3, and IAP5), which did not
produce any detectable TH activity at 0.15 mg/mL.
46
Carrot and
winter rye AFPs had a TH of 0.35 and 0.33 °C at relatively much
higher concentrations of 1 and 60 mg/mL, respectively.
30,47
Although at similar concentrations, carrot AFP had slightly
lower TH (0.14 °C) than seabuckthorn.
48
IAC purified PGIP was found to be N-glycosylated as iden-
tified by a gel shift from 41 to 39 kDa after glycosidase treat-
ment (Figure 8B). Antifreeze activity analysis with glycosidase
treated PGIP showed similar antifreeze activity, suggesting that
glycosylation is not required for antifreeze activity (Figure 8C).
To date, only three plant AFPs are known to be glycosylated,
out of which only one AFP from S. dulcamara requires glyco-
sylation for its activity.
49
The AFP detected in this study showed
glycosylation independent antifreeze activity as observed for
the other two glycosylated AFPs from carrot and Lolium.
49
Glycosylation of AFPs could have some other roles either in
targeting or in protein stabilization.
Sensitivity of purified antifreeze activity to proteinase-K
confirmed its proteinaceous nature (Figure 8C). Heat treat-
ment of PGIP resulted in complete loss of its activity, showing
its heat labile nature. However, heat stability studies on anti-
freeze activity in ECPs showed 32% of the activity to be
contributed by the heat stable proteins. The AFP purified in
this study (PGIP) is heat labile, suggesting existence of other
heat stable AFPs in secretome. Heat stable antifreeze proteins
have been previously identified from Lolium,
50
Deschampsia
antartctica,
51
carrot,
30
and winter wheat grass.
52
The end point of IRI (lowest concentration of AFPs that still
blocks the recrystallization) for PGIP was determined as 12 μg/mL
by its serial dilutions (Figure 8E). However, this IRI activity
exhibited by purified PGIP was comparatively lesser than other
AFPs purified from Lolium (3 μg/mL for IAP 3 and IAP 5, and
0.6 μg/mL for IAP 2),
46
carrot (1 μg/mL),
30
and Forsythia
suspensa (6 μg/mL).
53
Seabuckthorn PGIP showed least IRI activity in comparison
with other plant AFPs, while its TH is comparable or higher.
Yu et al. showed that IRI and TH activity are two independent
properties of AFPs, and there is no direct correlation between
the two.
9
Earlier, it was shown that Lolium AFPs (IAP 2, IAP 3,
and IAP 5), which showed maximum IRI activity, did not have
any detectable TH activity.
46
Characterization of seabuckthorn PGIP showed a weak TH
activity, which is a characteristic of plant AFPs. However,
during purification, no significant increase in the TH activity
was observed from total ECPs (0.13 °C) to the purified PGIP
(0.19 °C). Similar results were observed in S. dulcamara
17
and
carrot.
30
Smallwood et al. suggested that this may be due to the
absence of a cofactor required for the antifreeze activity, which
is probably lost during the purification.
30
■CONCLUSIONS
This is the first report of LT induced secretome analysis
in plants using a 2-DE-MS approach. LT stress changed the
abundance of 25% ECPs. On the basis of the functions of the
freeze induced proteins, a LT induced signaling network is
proposed (Figure S3 (SI)).
To the best of our knowledge, this is also the first report of
antifreeze activity analysis in seabuckthorn. A 41 kDa glycosyl-
ated, heat labile protein, identified as PGIP, was purified. High
degree of similarity (93%) of seabuckthorn and carrot AFPs
suggests a common function/role for both.
Sharing of 18% LT responsive proteins in secretome with
other subproteomes (mitochondria, plastids, nucleus, and plasma
membrane) confirmed that the identified proteins are indeed LT
responsive (Table S1 (SI)). Interestingly, defense/stress related
proteins were unique to the secretome except thaumatin like
protein and phenylalanine ammonia lyase, which were also
identified in the plasma membrane proteome. Chitinase, one of
the defense related proteins, showed 60-fold effective induction
after freezing stress, validating relevance of this defense related
protein in LT stress in secretome. Enzymes involved in the ROS
metabolism were identified in the secretome as well as in the
plastidome, suggesting that antioxidant battery of enzymes would
participate in cold stress signaling in both of the compartments.
This was further supported by 25.6-fold effective induction of
SOD by freezing stress in the secretome. Calmodulin-1, a
calcium sensor, was another interesting target identified in the
nuclear proteome, also suggesting similarity and continuity in
calcium mediated LT signaling between the two organelles. Cold
induced nuclear proteome had highest percentage of signaling
components, followed by secretome and plasma membrane,
suggesting their active participation in LT signaling. Surprisingly,
these were absent in plastid and mitochondria, indicating
relatively insignificant retrosignaling in LT.
Overall, these observations suggest the secretome to be a
dynamic player in LT signaling involving different proteins,
including antifreeze proteins. We expect that these results will
provide a better understanding of the involvement of secretome
in LT signaling in plants. Our future efforts would be to purify
and catalogue the complete repertoire of AFPs from H. rhamnoides
and other two species, H. salicifolia and H. tibetana,presentin
India. Few efficient candidates would be exploited for their
agricultural as well as biomedical applications.
■ASSOCIATED CONTENT
*
SSupporting Information
Figure S1: Optimization of seabuckthorn seed germination and
growth procedures. Figure S2: Functional categorization of cold
induced identified proteins in secretome. Figure S3: A putative
signaling network of LT induced proteins in shoots secretome.
Figure S4: Phylogenetic tree of polygalacturonase inhibitor
protein (PGIP) identified from different organisms. Figure S5:
Histograms of the functional classifications of the cold responsive
proteins identified in different organelles. Table S1: A comparative
analysis of cold induced targets with targets identified in other
subproteomes. This material is available free of charge via the
Internet at http://pubs.acs.org.
■AUTHOR INFORMATION
Corresponding Author
*E-mail: rdeswal@botany.du.ac.in. Tel./Fax: 91-011-27662273.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
This work was supported by a grant (BT/PR10799/NDB/51/
171/2008) from Department of Biotechnology, Government of
Journal of Proteome Research Article
dx.doi.org/10.1021/pr200944z |J. Proteome Res. 2012, 11, 2684−26962694
India and a research grant provided by University of Delhi to
R.D. R.G. thanks DBT for a research fellowship.
■ABBREVIATIONS
LT, low temperature; CA, cold acclimation; SZA, subzero
acclimation; AFPs, antifreeze proteins; REL, relative electrolyte
leakage; G6PDH, glucose-6-phosphate dehydrogenase; MS,
mass spectrometry; TH, thermal hysteresis; IAC, ice adsorption
chromatography; IRI, ice recrystallization inhibition; ECPs,
extra cellular proteins; PGIP, polygalacaturonase inhibitor
protein
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