isolation and characterization of cold stress...
TRANSCRIPT
CHAPTER 2
ISOLATION AND CHARACTERIZATION OF COLD STRESS
INDUCIBLE GENES IN CARROT BY SUPPRESSION SUBTRACTIVE
HYBRIDIZATION (SSH)
2.0 ABSTRACT
Daucus carota is cultivated widely in tropical and temperate regions, but grows
best in cool climates. Suppression Subtractive Hybridization (SSH) is a PCR based
method used to selectively amplify differentially expressed cDNAs and simultaneously
suppress non-target cDNAs. A subtraction forward library was constructed using total
RNA isolated from the leaves of cold stressed carrot plants to determine the genes
upregulated during cold stress. Out of the hundreds of clones obtained, randomly selected
clones were sequenced. From these sequences, 43 promising clones were submitted to the
NCBI EST database. Sequence analyses revealed the functions of these genes, which
were related to signal transduction, osmolyte synthesis and transport, regulation of
transcription, translation and protein folding. Sq RT-PCR analyses of Dc cyclin, Dc WD
and Dc profilin showed that, the first 2 genes were upregulated, where as Dc profilin
showed constitutive expression. However, the SSH analysis showed that all the 3 genes
were upregulated, as it is a much more sensitive technique.
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2.1 INTRODUCTION
A variety of molecular biological techniques are available to study the global gene
expression levels between two mRNA populations and to identify the differentially
expressed transcripts (Munir et al., 2004). The routine techniques used for the differential
expression studies are, representational difference analysis (RDA) (Lisitsyn et al., 1993),
Differential Display RT-PCR (DD RT-PCR) (Liang & Pardee 1992), Serial Analysis of
Gene Expression (SAGE) (Velculescu et al., 1995), Microarrays (Park et al., 2004) and
Suppression Subtractive Hybridization (SSH) (Diatchenko et al., 1996).
Suppression Subtractive hybridization (SSH) is a valuable tool for identifying the
differentially regulated genes, which are important for growth and differentiation of cells.
Many subtractive hybridization techniques have been developed in the last decade and
were used to isolate many genes from different systems (Sargent & Dawid 1983;
Hedrick et al., 1984; Hara et al., 1991; Wang & Brown 1991; Hubank & Schatz 1994).
Eventhough there are some advantages for each method, many of them require tedious
procedures, more amounts of starting material and are not cost effective, thereby reducing
their overall utility. SSH is a widely used technique to separate DNA molecules that
distinguishes two closely related DNA samples. The major applications of SSH are in
cDNA subtraction and genomic DNA subtraction studies. SSH is one of the common
technique for generating subtracted cDNA or genomic DNA libraries (Lukyanov et al., 1994;
Diatchenko et al., 1996; Gurskaya et al., 1996; Akopyants et al., 1998). In SSH, the
normalization step eliminates the intermediate steps of physical separation of single
stranded and double stranded cDNA molecules and requires only single round of
hybridization reaction, which results 1,000 fold enrichment of the differentially expressed
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genes (Rebrikov et al., 2000). The subtraction process is based on suppression PCR effect and
it combines subtraction and normalization reaction in a single procedure. The normalization
step in subtraction hybridisation equalizes the abundance of different cDNA fragments in a
single round of subtraction (Diatchenko et al., 1996; Gurskaya et al., 1996; Jin et al., 1997).
However, the enrichment level of a particular cDNA depends on factors like, the original
abundance of particular cDNA, the ratio of its concentration in the samples used for
subtraction and the concentration of other differentially expressed cDNAs.
2.1.1 Principle of SSH- As the name indicates, the process includes two different
techniques in the isolation of uniquely expressed genes. The cDNA population in which
specific transcripts are to be found is called tester cDNA for the subtraction reaction and
the reference cDNA sample population for the subtraction reaction is called driver cDNA
(Diatchenko et al., 1996). The tester cDNAs and the driver cDNAs are digested with a
4 base cutters like RsaI or AluI to make blunt end cDNAs (Rebrikov et al., 2000).
The tester cDNA is then subdivided into two fractions (1 and 2) and each one is separately
ligated to different double stranded adapter molecules (adapters 1 and 2R respectively).
The ends of the synthetic oligonucleotides are designed in such a way that, they are not
phosphorylated at the 5‘end, so only one strand of each adapter becomes covalently
attached to the 5' end of the cDNAs. The samples are then heat denatured and allowed to
anneal with denatured driver cDNA population. During the first hybridization step, the
subset of single stranded tester molecules is normalized and hence concentrations of high
and low abundance transcripts become roughly equal. The normalization process occurs
because the annealing process generating homohybrid and heterohybrid cDNAs are more
rapid for more abundant molecules, (due to the second order of hybridization kinetics)
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than less abundant cDNA, which remains single, stranded. By controlling the extent of
the hybridization reaction, the single stranded forms of highly abundant cDNAs can be
reduced to the same levels as those of less abundant cDNA populations, thereby
normalizing the representation of tester cDNA population (Hames & Higgins 1985).
At the same time, the population of type A molecules (fig. 2.1) is significantly enriched
for differentially expressed cDNA population, because it is common for tester and driver
samples non-target cDNAs known as type C molecules with the driver cDNA. During the
second step of hybridization, the two samples from the first set of hybridization reaction
are mixed and annealed further with additional freshly denatured driver. Under these
conditions, only single stranded A type tester cDNAs are able to re-associate and form
(B), (C), and new (E) hybrids. Type E hybrids are double stranded (ds) tracer molecules
with different ss ends, one of which corresponds to Adapter1 and another to Adapter 2R.
The entire cDNA population is then subjected to two rounds of PCR to selectively
amplify the differentially expressed sequences (Rebrikov et al., 2000). The first PCR is
performed with adapter specific primers. This ensures that those cDNAs with both the
adapter sequences are only amplified. Type A and D cDNA population lack primer
annealing sites and cannot be amplified. Type B cDNA population form stem-loop like
structures that suppress PCR amplification (Lukyanov et al., 1994; Siebert et al., 1995).
Type C cDNA population have only one primer-annealing site, hence it can be amplified
only at a linear rate. Only type E cDNA population, which have different adapter
sequences at their ends having two different primer annealing sites that can be amplified
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exponentially. A nested PCR is followed by the first PCR to increase the specificity of
the reaction. The differentially expressed cDNA sequences are greatly enriched in type E
fraction from the subtracted cDNA pool.
Figure 2.1 – SSH Principle (Clonetech Laboratories, CA, USA)
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SSH was used to study differentially expressed transcripts in Arabidopsis with
response to ozone, bacterial and oomycete pathogens and the signalling compounds such
as salicylic acid (SA) and Jasmonic acid (Mahalingam et al., 2003). A substracted cDNA
library was constructed in the high altitude plant Lepidium latifolium to screen the cold
responsive genes (Aslam et al., 2009). A novel metallothionein like gene was identified
from the subtracted library of Cicer microphyllum, which responds to various abiotic
stresses (Singh et al., 2010). Kang et al., (2010) identified several genes by SSH, which
were induced by salt stress in seedlings of Medicago trunculata. An AP2 containing
protein was involved in response to metal stress in Physcomitrella patens was identified
by a modified SSH protocol (Cho et al., 2007). Xu et al., (2006) identified several
pathogen induced defense genes in Chestnut rose after constructing a cDNA library by
SSH. Gene expression in Fucus vesiculosus L. was investigated using cDNA library
generated by SSH for algae undergoing mild desiccation stress and identified many genes
upregulated (Pearson et al., 2001). A SSH library was constructed in Mitragyna speciosa
to identify the expression of genes involved in secondary metabolism after treatment with
Salicylic acid (Jumali et al., 2011).
In this study, a forward cDNA library was constructed to identify the cold
responsive genes in the vegetative tissues of Daucus carota.
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2.2 MATERIALS AND METHODS
2.2.1 Plant Material, Growth Conditions and Cold Stress Treatment- D.carota cv Kuroda
seeds were procured from the Horticulture training centre, Ooty, India. The seeds were sowed
(in commercially available soil mix in pots) in the containment facility of Defence Institute of
Bio-Energy Research, Haldwani, India. The germinated plants were maintained in controlled
conditions at 25°C with 16/8h light/dark cycle. The plants were nourished with 1/10 strength
sterile MS salt solution. One month old plants were exposed to cold stress at 4°C for 24h in a
cooling incubator with 16/8h light/dark cycle. Plants maintained in the containment facility
with normal conditions were used as control for the experiment.
2.2.2 DEPC Treatment- Diethylpyro Carbonate (DEPC) is a strong inhibitor of RNases
that works by covalently modifying RNases. All the glasswares used for the RNA related
work were thoroughly rinsed with detergent and then with sterile distilled water.
The glasswares were baked in hot air oven at 240°C overnight and then immersed in 0.1%
DEPC solution overnight. The glasswares were autoclaved at 121°C for 1h. The
microfuge tubes and pipetteman tips used were of nuclease free polypropylene (sterile).
The non-disposable plastic wares were thoroughly rinsed with 0.1N NaOH, 1mM EDTA
followed by nuclease free water. Polycarbonate or polystyrene materials were cleaned by
immersing in 3% hydrogen peroxide solution for 10 min. Peroxide solution was removed
by rinsing with DEPC treated and with sterile water prior to use. Electrophoresis tanks,
gel casting trays and combs were cleaned with detergent solution (e.g., 0.5% SDS)
thoroughly rinsed with RNase-free water and was allowed to dry. All the solutions
(except Tris based solution as DEPC interferes with the activity of Tris) were
made/diluted using 0.1% DEPC.
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2.2.2.1 RNA Isolation from Control and Cold Treated Plants- Young leaves from the
control and cold stress treated plants were harvested at the same time (in order to avoid
any physiological variation) and immediately frozen in liquid Nitrogen until further
process. The total RNA was isolated from control and test sample using Qiagen RNeasy
RNA isolation kit (Qiagen, Hilden, Germany) as per the manufacturer‘s instructions.
The leaves (approximately 100mg) were ground well with liquid Nitrogen. The powdered
leaves were then transferred to RNase free tube and 450µL of RLT buffer (containing
45µL of β-mercaptoethanol) were added and vortexed vigorously. The lysate was
transferred to a QlA shredder column (lilac) in a 2mL collection tube. The samples were
then centrifuged at 12000g for 2 min. Absolute ethanol (225µL) was added to the lysate
and mixed immediately. The samples were then transferred to an RNeasy mini column
(pink) that is placed in a 2mL collection tube and centrifuged for 15s at 12000g. Buffer
RW1 (700µL) was added to the RNeasy column and the samples were centrifuged for 15s
at 12000g. On column DNase digestion was performed to remove the genomic DNA
contaminants from RNA. DNase (30µL) was mixed with 70µL of RDD buffer and added
directly on to the membrane of the column. The samples were incubated at RT for
15 min. After the digestion, the column was washed with 500µL of RW1 buffer. To the
column, 500µL of RPE solution was added and centrifuged for 15s at 12000g twice.
The RNA was eluted by adding 40µL RNase free water to the column and centrifuged for
1 min at 12000g. The concentration of RNA was quantified and the integrity of RNA was
checked in formamide gel.
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2.2.2.2 Purification of mRNA from Total RNA
2.2.2.2.a- Annealing of Probe- The mRNA was purified from the total RNA using
PolyATtract®
mRNA isolation system I (Promega, Madison, USA) according to
manufacturer‘s instruction. The RNA from the control and cold treated samples were
taken separately in an RNase free tube and the volume was made upto 2.43mL using
RNase free water. The tubes were placed in a heating block for 10 min at 72°C to
denature the secondary structure of RNA. Biotinylated oilgo dT (10µL) and 60µL of 20X
SSC were added to the tube and mixed gently by pipetting back and forth. The contents
were allowed to cool at RT.
2.2.2.2.b- Washing of Streptavidin-Paramagnetic Particles- The Streptavidin
MagneSphere® Paramagnetic Particles (SA-PMP‘s) requires BSA for stabilization,
which is present in the storage buffer. The SA-PMPs was provided at a concentration of
1mg/mL in PBS, 1mg/ml BSA and 0.02% Sodium azide. The tubes with SA-PMPs were
resuspended (one tube per sample) by flicking the bottom of the tube gently until they
were completely dispersed and captured by placing the tube in the magnetic stand, until
the SA-PMPs collected from the side of the tube. The supernatant was carefully removed
and the particles were washed thrice with equal volume of 0.5X SSC.
2.2.2.2.c- Capture and Washing of Annealed Oligo (dT)-mRNA Hybrids- The entire
content of the reaction mix was carefully transferred to the tube containing the SA-PMPs
and incubated at room temperature for 10 min. The tubes were inverted in every 2 min to
ensure proper mixing of RNA and oligo dT. The tubes were then placed in the magnetic
stand and the supernatant was carefully removed without disturbing the SA-PMP
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particles. The particles were washed 4 times with 1.5mL of 0.1% SSC by gently flicking
the bottom of the tube until all particles were resuspended. The supernatant was removed
after the final wash (without disturbing the pellet).
2.2.2.2.d- Elution of mRNA- Finally SA-PMP particles were added to the 50µL of
RNase free water and gently resuspended by flicking the tube. The tubes were then
placed in the magnetic stand and the eluted mRNA was carefully separated from the
particles and transferred to a fresh RNase free microfuge. The microfuge was centrifuged
at 12000g for 1 min to pellet the magnetic particles and mRNA was transferred to a fresh
tube.
2.2.2.3 First Strand cDNA Synthesis - In a RNase free PCR tube, 2µg of poly A mRNA
and 1µL (10µM) oligo dT were added (for both tester and driver separately) and the
volume was made to 5µL using nuclease free water. The contents were briefly mixed and
incubated at 72°C for 2 min in a thermal cycler. The samples were snap cooled in ice
after the incubation period. To the tube 2µL of 5X RT buffer (Fermentas Inc., Maryland,
USA), 1µL of 10mM dNTP mix (Fermentas Inc., Maryland, USA) and 1µL of 20U/µL
M-MLV RT (Fermentas Inc., Maryland, USA) were added and the volume were made to
10µL using nuclease free water. The contents in the tube were briefly mixed in a mini
centrifuge. Revere transcription was performed at 42ºC for 60 min in a thermal cycler.
The tubes were immediately placed in ice to terminate the reaction (after the incubation
period) and samples were used for the synthesis of cDNA.
2.2.2.4 Second Strand cDNA Synthesis- The whole sample from the first strand cDNA
was used for the synthesis of second strand. In the same tube, 48.4µL of nuclease free
water, 16µL of 5X second strand synthesis buffer, 1.6µL of dNTPs (10mM each) and
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4.0μL of 20X second strand enzyme were added. The final volume of the mix was made
to 80µL and incubated at 16°C for 2h in a thermal cycler. After the incubation period,
2μL (6 U) of Platinum Pfu DNA polymerase (Fermentas Inc., Maryland, USA) was
added and the contents were mixed well. The PCR was performed with the following
conditions, an initial denaturation at 95ºC for 3 min followed by 30 cycles of denaturation
at 95ºC for 15 s and extension at 66 ºC for 6 min. The reaction was terminated using 4μL
of EDTA/Glycogen mix. The sample was mixed thoroughly with equal volume of
Phenol: Chloroform: Isoamyl alcohol (25:24:1) and centrifuged at 12000g for 10 min at
RT. To the extracted upper aqueous layer, 25μL 4M NH4OAc and 187.5μL absolute
ethanol were added. The samples were then centrifuged at 14000g for 10 min at room
temperature, supernatant was removed, pellet was washed with 70% ethanol and air dried
and dissolved in 20μL sterile water.
2.2.3 RsaI Digestion- The purified product (second strand DNA) was used for the
digestion with RsaI (Fermentas Inc., Maryland, USA). Each tester (stress sample) and
driver (control sample) double stranded cDNA was digested with RsaI to generate
shorter, blunt ended ds-cDNA fragments that are optimal for subtraction and adapter
ligation. In a sterile PCR tube, 40μL of the purified PCR product, 5μL of 10X RsaI
buffer, 8μL of nuclease free water and 2μL (10U/μL) of RsaI were added. The samples
were briefly centrifuged and incubated in water bath for 2h at 37°C. The digestion
reaction was arrested using 4μL of 20X EDTA. The digestion products were purified
(as mentioned above) and used for adaptor ligation.
2.2.4 Suppressive Subtractive Hybridization and Construction of Subtracted cDNA
library- Suppressive Subtractive Hybridization (SSH) was carried out using Polymerase
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Chain Reaction (PCR) Select cDNA Subtraction kit (Clonetech laboratories, CA, USA)
according to manufacturer‘s instruction. Double stranded cDNA was synthesized from
the 2μg of poly A+ purified mRNA.
2.2.4.1 Ligation of Adaptor to Tester cDNA Population- The purified RsaI digested
cDNA was used for adaptor ligation. The purified cDNA (tester) was diluted in nuclease
free water (1μL cDNA is mixed with 4μL of sterile water) and the samples were kept in
ice. A ligation mix was made as shown in table 2.1,
Sl. No Component Volume per
reaction
01 Nuclease free water 3μL
02 5X ligation buffer 2μL
03 T4 DNA ligase (400U/ μL) with 3mM ATP (final ATP
concentration in mix will be 300 μM).
1μL
Table 2.1- Ligation Mix for Adaptor Ligation 1.
The ligation mix was used for preparing the ligation reaction with two different adaptors
as mentioned in table 2.2,
Sl. No Component Tester
1-1
Tester
1-2
01 Diluted tester cDNA 2μL 2μL
02 Adaptor 1 (10 μM) 1μL -
03 Adaptor 2R (10 μM) - 1μL
04 Ligation master mix 7 μL 7 μL
Final Volume 10μL 10μL
Table 2.2- Ligation Mix for Adaptor Ligation 2
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The components were briefly centrifuged and incubated in a thermal cycler for
overnight at 16°C. One microliter of EDTA was added to arrest the reaction and the
samples were incubated at 72°C for 5 min to inactivate the ligase.
2.2.4.2 First Hybridization Reaction- In the first hybridization, an excess of driver
cDNA was added to tester cDNA (ligated with adaptor) samples were then heat
denatured and allowed to anneal. The remaining ss cDNAs (which were available for the
second hybridization) was dramatically enriched for differentially expressed sequences
as non-target cDNAs present in the tester and driver cDNA forming hybrids.
The hybridization buffer was made to thaw in RT and hybridization mix was made
according to the following (table 2.3)
Sl. No Component Tester
1-1
Tester
1-2
01 RsaI digested Driver cDNA (excess) 1.5μL 1.5μL
02 Adaptor 1-ligated Tester 1-1 1.5μL -
03 Adaptor 2R-ligated Tester 1-2 - 1.5μL
04 4X Hybridization Buffer 1μL 1μL
Final Volume 4μL 4μL
Table 2.3- First Hybridization Mix Components
The samples were overlaid with a drop of mineral oil and were briefly centrifuged.
The samples were initially incubated at 95°C for 2 min and left for hybridization at 68°C
for 12h.
2.2.4.3 Second Hybridization Reaction- The two samples from the first hybridization
reaction were mixed together and freshly denatured driver cDNA was added to further
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enrich differentially expressed genes and for the formation of new hybrid molecules that
consists differentially expressed cDNAs with different adaptors on either ends.
The hybridization mix for the second round of hybridization was made as mentioned in
table 2.4,
Sl. No Component Volume per reaction
01 Driver cDNA 1μL
02 4X Hybridization Buffer 1μL
03 Nuclease free water 2μL
Final volume 4μL
Table 2.4- Second Hybridization Mix Components
The samples were briefly mixed by centrifugation and overlaid with 1μL of mineral
oil. The sample was denatured at 98°C for 2 min in a thermal cycler. The following step is
important to ensure that both the hybridization samples mixed with the denatured driver
at the same time. The pipette tip was gently touched to the mineral oil/sample interface of
the tube containing hybridization sample 2 and the entire sample was drawn from the
tube. The tip from the tube was removed and a small amount of air was drawn into the tip
creating, a slight air space below the droplet of sample. The same procedure was repeated
to take the freshly denatured driver cDNA in the same tip. At the end, the tip contained
both the hybridized sample 2 and the freshly denatured driver cDNA. The entire sample
was transferred to the tube containing first hybridization mix and it was mixed
immediately by pipetting up and down. The samples were centrifuged and left for second
hybridisation at 16°C for overnight in a thermal cycler. At the end of the hybridization
the samples were diluted using 200μL of dilution buffer and incubated at 68°C for 7 min.
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2.2.5 Amplification of cDNA Inserts by PCR and Nested PCR- The resultant
subtractive product was amplified by PCR using primers that were complimentary to
sequence of adapters 1 and 2R. The diluted cDNA was used as the template for the first
PCR reaction. The PCR mix was made as follows (table 2.5)
Sl. No Component Volume per reaction
01 Nuclease free water 18.5μL
02 Taq buffer 10X 2.5μL
03 dNTPs (10mM) 1μL
04 Diluted template 1μL
05 PCR primer 1 (10μM) 1μL
06 Platinum Taq DNA polymerase (2.5 U/μL) 1μL
Final Volume 25μL
Table 2.5- Components of Master Mix for First PCR
The samples were briefly centrifuged and overlaid with mineral oil. The reaction
mixture were incubated at 75°C for 5 min in a thermal cycler to extent the adaptors and
the PCR reaction was immediately commenced. The cycling conditions for the first PCR
was as follows, denaturation at 94°C for 30s followed by annealing at 66°C for 30s and
extension at 72°C for 90s for 27 cycles. At the end of the PCR, 8μL of the PCR product
was analyzed in agarose gel. The PCR product from the first reaction was diluted in water
in the ratio of 1:9 (3μL of the PCR product with 27μL of nuclease free water) and was
used as the template for the second reaction with nested primers.
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A master mix for the second PCR was made as follows table 2.6,
Sl. No Component Volume per reaction
01 Nuclease free water 17.5μL
02 Taq buffer 10X 2.5μL
03 dNTPs (10mM) 1μL
04 Diluted template 1μL
05 Nested PCR primer 1 (10μM) 1μL
06 Nested PCR primer 2R (10μM) 1μL
07 Platinum Taq DNA polymerase (2.5 U/μL) 1μL
Final Volume 25μL
Table 2.6- Components of Master Mix for Nested PCR
The samples were briefly centrifuged and overlaid with mineral oil. The cycling
conditions for nested PCR was as follows, at 94°C for 30s followed by annealing at 66°C
for 30s and extension at 72°C for 90 s for 12 cycles. PCR product (8μL) was analyzed in
agarose gel.
2.2.6 Transformation of the Subtracted Library to E.coli
2.2.6.1 RsaI Digestion of Subtracted Library and Purification- The subtracted cDNA
library product was purified using Genei PureTM
Quick PCR purification kit, GeneITM
,
India. The purified product was digested with RsaI. In a sterile PCR tube, 20μL of the
purified PCR product (subtracted forward cDNA library), 2.5μL of 10X RsaI buffer,
1.5μL of nuclease free water and 1μL (10U/μL) of RsaI were added. The samples were
briefly centrifuged and incubated in thermal cycler for 2h for digestion. The digested
product was purified and eluted in a final volume of 10μL twice.
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2.2.6.2 Isolation of Plasmid from E.coli Harbouring pBluescript KS- A single colony
of E.coli. was inoculated in 50mL of LB containing 50μL of Ampicillin (100mg/mL) and
incubated overnight at 37°C in orbital shaker. The overnight grown culture was used for
the isolation of plasmid DNA (pBS). The plasmid DNA was used for restriction enzyme
digestion.
2.2.6.3 Digestion of the pBS Plasmid with SmaI- The pBS plasmid was digested with
SmaI enzyme inorder to obtain blunt ends. The digestion mix contained plasmid DNA
20μL, 10X SmaI digestion buffer (wih BSA) 2.5μL, nuclease free water 1.5μL and SmaI
1μL (10U/μL). The samples were briefly centrifuged and incubated at 37°C in a thermal
cycler for 3h.
2.2.6.4 Cloning of Subtracted cDNA Library to pBS Plasmid- The digested cDNA
library was cloned to linearised pBS plasmid by blunt end ligation. The components of
the ligation mix were made as shown in table 2.7.
Sl. No Component Volume per
reaction
01 Digested plasmid DNA (200ng) 2μL
02 Digested cDNA 2μL
03 5X ligation buffer 2μL
04 Nuclease free water 3μL
05 T4 DNA ligase (400U/ μL)
(with 3mM ATP, final ATP concentration in mix will
be 300μM)
1μL
Total volume 10μL
Table 2.7- Components of the Ligation Mix
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The contents were briefly mixed and centrifuged. The samples were incubated in
a thermal cycler overnight at 16°C. Ligated product (2μL) was used for transformation to
E.coli Dh5α.
2.2.6.5- Transformation of Ligated Product to E. coli - The ligated product was
transformed into E. coli Dh5α cells (Genotype: F Φ80lacZΔM15 Δ(lacZYAargF) U169
recA1 endA1 hsdR17 (rk , mk ) phoA supE44 thi-1 gyrA96 relA1 tonA). The E. coli
competence cells were prepared according to Sambrook & Russell (2001) with minor
modification. LB medium (Himedia Laboratories, India) was routinely used to culture
E. coli. Single colony was inoculated in 25mL LB and incubated at 37°C for 6h.
Inoculum (1%) was transferred to 50mL LB and incubated in an orbital shaker until it
reaches 0.5-0.6 OD600. The culture was chilled in ice for 30 min. The cells were pelleted
by centrifugation at 1500g for 10 min at 4°C. The pellet was dispensed in 4mL of ice cold
sterile 0.1M CaCl2 and centrifuged at 1500g for 10 min at 4°C. The cells were finally
resuspended in 1mL 0.1 M CaCl2 and 50μL aliquots were made and stored at -80°C until
transformation.
The 50µL aliquots of competence cells stored at -80°C were thawed using ice for
30 min and ligated product (5µL) was directly pipetted over competent cells. The cells
were mixed gently by tapping 4-5 times, incubated on ice for 30 min, which was
followed by a heat shock treatment at 42°C for 60 s. After the heat shock, the cells were
immediately placed in ice for 2 min. SOC media (250µL) was added to the tubes.
The tubes were finally incubated at 37°C for 1h at 225 rpm in orbital shaker. The cultures
were appropriately diluted in LB medium and 50-100µL of each culture was spread on
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selection media (LB agar supplemented with 50mg/L Ampicillin). Isopropyl β-D Thio
Galactoside (8µL of 0.2 M IPTG) and 40µL of 20mg/mL X-Gal (5-bromo 4-chlororo
3-indolyl β- D galactoside) were spread on the plates prior to plating of the transformed
cells for blue/white selection to identify the recombinant clones. The plates were sealed
with parafilm and incubated upside down at 37°C overnight.
2.2.7 Differential Screening of the Subtracted Clones- The white transformed colonies from
the subtracted library was screened by colony PCR using M13 universal primers. The colonies
were patch streaked in fresh LB agar plates with 100mg/L Ampicillin and the tip containing the
colony was used as the template source for PCR. The PCR mix contained, 5μL 10X Taq
buffer, 2.5μL MgCl2 (25 mM), 1μL dNTP mix (10 mM each), 1μL each of M13 forward
primer (5‘-GTA AAA CGA CGG CCA GT-3‘) and M13 reverse primer (5‘-AAC AGC TAT
GAC CAT G-3‘) (10 pM), 39.3μL of PCR grade water and 0.2μL Taq DNA polymerase
(5U/μL). PCR was carried out with an initial denaturation at 95°C for 5 min, followed by
30 cycles of 95°C for 30s, 55°C for 60s, 72°C for 90s and a final extension of 5 min at 72°C.
The PCR products were resolved in 2% agarose gel and stained with ethidium bromide.
2.2.8 Plasmid Isolation and Sequencing of the Subtractive Clones- Clones were
selected randomly and inoculated in LB broth containing 50mg/L Ampicillin. The tubes
were incubated at 37°C for overnight in an orbital shaker and the plasmid DNA was
isolated from the selected clones. The plasmid DNA was sequenced in Chromous
Biotech, Bangalore, India using M13 forward primer.
2.2.9 EST Sequence Analysis - The vector sequences were first removed from the
obtained sequence using the Vec Screen online software (www.ncbi.nlm.nih.gov/
VecScreen/VecScreen.html). The EST sequences were analyzed for homology using
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Basic Local Alignment Search Tool (BLASTN) from the National Center for
Biotechnology Information database (www.ncbi.nlm.nih.gov/blast/). Homology searches
were performed against non-redundant nucleotide sequence using BLASTN of BLAST
programme. The unique EST sequences were submitted in the EST GenBank database.
2.2.10 Semi quantitative (sq) RT-PCR Analysis- The transcript accumulation of
3 genes was studied after giving cold stress to carrot plants. One month old carrot plants
were given stress for 5 days at 4°C. Samples were then harvested every 24h and were
immediately frozen in liquid nitrogen and stored in -80°C until further process. RNA was
isolated from all the samples using Qiagen RNeasy RNA isolation kit (Qiagen, Hilden,
Germany) and on column DNase digestion was performed to remove the genomic DNA
from the RNA sample as mentioned in the section 2.2.2.1. The RNA was quantified in
Nanodrop (Thermo Scientific, USA) and the integrity was checked in formamide gel.
RNA (2µg) was used for the synthesis of cDNA using RETROscript® Kit- Reverse
Transcription for RT-PCR (Ambion Inc, USA) according to manufacturer‘s instruction.
Primers were designed for Dc WD protein, Dc Profilin 4 and Dc Cyclin 2b. The first
strand cDNA was diluted 10 times using nuclease free water and was used as template for
PCR. PCR was performed using Ready to go PCR beads (GE healthcare, NJ, USA).
The primer sequences are shown in the table 2.8. Dc Elongation factor 1 alpha (EF1α)
was used as the internal control for sqRT PCR. The PCR cycle consists of 28 cycles with
an initial denaturation at 94°C for 5 min followed by denaturation at 94°C for 30s,
annealing at 60°C for 30s and extension at 72°C for 30s. A final extension was 72°C for
10 min. The PCR products were separated in 2% agarose gel and stained with ethidium
bromide (80ng/μL). The sq RT-PCR was repeated twice to verify the results.
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Sl. No Gene Primers
01 Dc WD Fw: 5‘- TGT CAA TGG CCT CCA CAA ATT-3‘
Rv: 5‘- TTT ACA GCT GAA GTG TGT TCT TCC A-3‘
02 Dc Profilin 4 Fw: 5‘- AAG CTC TGG TGT TTG GAG T-3‘
Rev: 5‘- TAA TCT CCA AGC CTC TCA AC-3‘
03 Dc Cyclin 2b Fw: 5‘- ACA ATT CGA GTG CTG TTT CT-3‘
Rev: 5‘-CTG TGG GTC ATC AAA TTT CT-3‘
04 Dc EF1α Fw: 5´- TGG TGA TGC TGG TTT CGT TAA G -3´
Rev: 5‘-ATG GGA GGG TAG GAC ATG AAG GT -3´
Table 2.8- Primers used for the sq RT-PCR Analysis
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2.3 RESULTS AND DISCUSSION
2.3.1 Construction of Subtraction cDNA Library- The young rosette of the carrot
leaves of control and cold stressed plants were used for RNA isolation. The reliability of
the SSH method mainly depends on the quality and quantity of RNA used for the
subtraction procedure. The clear bands of both the 18S and 28S rRNA in the formamide
gel showed that the RNA is intact (fig. 2.2). High quality of mRNA was separated from
the total RNA and was used for the synthesis of cDNA. The forward library was
constructed using the stress-induced sample as the tester population and the control
sample as the driver population to determine the genes upregulated during cold stress.
The cDNAs samples, which were differentially expressed, were obtained after two
rounds of hybridization procedure followed by two rounds of suppression PCR (fig. 2.3).
The cDNAs thus obtained were enriched with genes upregulated during cold stress.
Figure 2.2 RNA Isolated from Control and Cold Stressed Carrot Plant
Lane M- Molecular weight marker; Lane 1- RNA from control plant;
Lane 2- RNA from cold stressed carrot
28S rRNA
18S rRNA
M 1 2
250bp
5 kb
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Figure 2.3- Result of SSH using cDNA from Cold Stressed Leaves as Tester and
Control Leaves as Driver.
Lane M- Molecular weight marker, Lane 1 & 2- Smear after first round of PCR
Lane 3 & 4- Forward subtracted library
Figure 2.4- Screening of Insert Size by Colony PCR using M13 Primers
Lane M- DNA ladder
Lane 1-15 (upper panel) and lane 1- 19 (lower panel) - clones from forward library
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
250bp
500bp
10kb
10kb
250bp
500bp
M 1 2 3 4
Subtracted forward library
250bp
10kb
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2.3.2 Cloning and Differential Screening of the Subtracted cDNA Library- The PCR
product of the second round of PCR reaction that represented the subtracted cDNA
library was ligated to pBlue script KS (+) vector and transformed to E. coli cells.
The white colonies were screened using M13 primers and randomly selected clones were
used for sequencing (fig. 2.4)
2.3.3 Sequence Analysis- Around 75 randomly selected clones were sequenced. Around,
62 clones produced readable sequences. After removing the redundant or vector
backbone sequences 43 sequences of the potential clones were submitted in the public
EST database. The accession number of 43 potential clones with their putative function is
summarized in the table 2.9.
2.3.4 sq RT-PCR- The sq RT-PCR results indicate that cyclin and WD protein
upregulated after stress, whereas there was no change in expression level of profilin
(fig. 2.5). Cyclin is one of the major proteins that regulate different stages of the cell
cycle by its concerted expression and degradation. They along with cyclin dependent
kinases achieve by phosphorylating different targets. Interaction of Arabidopsis cyclin
D2 expressed in transgenic rice with endogenic cyclin-dependent kinase enhanced
seedling growth (Oh et al., 2008). Cell cycle activities involved in stress responses are
mediated by transcription factors (Morano et al., 1999). Transgenic rice expressing
OsMYB3R-2 enhanced low temperature tolerance that has been shown to be mediated by
alteration in cell cycle (Ma et al., 2009). The transcript level of cyclin D2, cyclin B2-2
and Cyclin Dependent Protein Kinase (CDPK) were upregulated during drought stress in
Arabidopsis, wheat and rice respectively (Kamal et al., 2010). In the present study, the
cyclin2b was found to be upregulated in carrot during cold stress.
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The protein with WD repeats play a key role in signal transduction, cytoskeletal
dynamics, ribosomal RNA biogenesis (Neer et al., 1994; Smith et al., 1999), cytokinesis,
apoptosis, floral development and meristem organisation (Nocker & Ludwig 2003).
They have been classified based on sequence similarity (Nocker & Ludwig 2003).
Recently, a WD 40 in B.napus (BnSWD1) was reported to play a major role during salt
stress (Lee et al., 2010). SNF1 kinase is a key enzyme in plant steroid biosynthesis and it
phosphorylates the 3-hydroxy-3methylglutaryl-CoA reductase. Its activity is regulated by
PRL1, a conserved nuclear WD-protein that is implicated in cold tolerance in
Arabidopsis (Bhalerao et al., 1999). It was 2-16 folds repressed in leaves/shoots of cold,
high-salinity stressed chickpea (Mantri et al., 2010); on the other hand it was upregulated
in carrot during low temperature stress, suggesting that it may have a role in cold stress
tolerance.
The expression of profilin was unaffected under cold stress as seen from the sq
RT-PCR results (fig. 2.5), although SSH analyses show upregulation (table 2.9). SSH is a
powerful technique that can detect even minor changes in transcript levels as in the
present case. Profilins are a group of low molecular weight ubiquitous actin binding
eukaryotic proteins, which are involved in the remodelling of actin cytoskeleton
(Huang et al., 1996) and also involved in various signalling cascades in yeast and plants
(Vojtek et al., 1991; Machesky et al. 1993). Swoboda et al., (2001) reported that
remodelling architecture of cytoplasm is a normal process in cells to maintain the
membrane integrity during various environmental stresses and in carrot profilins might
also be contributing to the cold tolerance.
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Figure 2.5- Expression Profile of 3 Genes of D. carota by sq RT- PCR (Dc Cyclin, Dc
WD and Dc Profilin)
Lane- C-Control sample, 1 to 5 Samples from 1st to 5
th day after stress.
2.3.5 Functional Classification of Cold Stress Induced ESTs- The unique ESTs were
annotated based on their similarities with existing sequences in GenBank using BLAST
based on their functions into 6 groups. They are cold, salt, drought, UV stress responsive
genes, genes involved in transcription/translation, genes involved in sugar/protein/lipid
metabolism, genes involved in maintaining structural integrity, genes involved in signal
transduction and genes of unknown functions (fig. 2.6).
C 1 2 3 4 5
RNA
Cyclin 2b
WD gene
Profilin
EF1α
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Fig 2.6- Grouping of Cold Responsive Gene Sequences based on their Functions
Derived from SSH Technique
2.3.6 Comparative Analysis of the Sequences with Cold Regulated Transcriptome of
Plants- A comparative analysis of the cold upregulated transcriptome was carried out, to
correlate and understand the mechanism of low temperature tolerance in plants.
The upregulation of several genes of varied functions suggests that, the unique ESTs may
play role in complex biological processes. Wang et al., (2007) have reported that all the
upregulated genes may not have a role in stress tolerance, but could be in response to
damages caused by the stress. The largest number of genes belongs to the groups that are
upregulated during cold, drought or salinity stress. Apart from this, the upregulation of
genes of unknown functions and those involved in transcription/translation suggests that
the responses to cold stress are rather complex and multigenic as it was reported by
(Sun et al., 2007; Zhang et al., 2009; Kang et al., 2010).
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There are several classes of transcription factors such as, AP2/ERF, DREB,
YABBY and Trihelix families, which are unique to plants and act as molecular switches
(Xie et al., 2009). Ramamoorthy et al., (2008) have identified that, some of these
transcriptional factors are involved in transcriptional regulation during environmental
stresses. Liu et al., (2011) reported that, the low osmotically responsive gene 2 (LOS 2)
from Poncirus trifoliate act as a transcriptional activator for various cold-responsive
genes. In carrot during cold stress expression of fas and DREB were upregulated. Fas is
involved in regulating flowering through YABBY in tomato (Cong et al., 2008). In carrot
normally vernalization is required for flowering and we propose that, the cold treatment
lead to the expression of fas, which in turn might initiate the flowering. The Dehydration
Responsive Element Binding (DREB) proteins have been found to confer tolerance to
cold/drought stress in Arabidopsis (Kasuga et al., 2004), wheat (Andeani et al., 2009),
rice (Dubouzet et al., 2003), cotton (Shan et al., 2007), and could be functioning similarly
in the carrot also. Similarly, the zinc finger CCCH protein enhanced in response to salt
stress in Arabidopsis (Sun et al., 2007). Other zinc finger proteins like Zat 12 and Gh ZFP 1
have been found to confer tolerance to oxidative stress and salt stress (Guo et al., 2009)
respectively.
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Sl.
No
Accession
Number Predicted gene family Putative Function
E-
value
1 GW316731 A. thaliana cyclin2b Regulator of cyclin dependent
kinases (Cell cycle regulator)
3e-33
2 GW342890 R. communis WD protein Signal transduction/cell cycle
regulation
5e-04
3 GW314857 Dc 4 profilin Actin binding protein 3e-24
4 GW343024/
GW343026
Solanum lycopersicum- FAS
protein
Encodes transcription factor 4e-12
5 GW315340 Chrysanthemum vestitum-
DREB
Cold tolerance 1e-15
6 GW342888 A. thaliana Zinc finger CCCH
protein
Cold/salt stress 2e-06
7 GW343025 Talaromyces stipitatus- Rop
GTPase activator
Cold/salt/drought stress 9e-06
8 GW316484 Dc RNA polymerase Transcription 7e-14
9 GW316488 Hypochaeris megapotamica-
maturase K
Intron splicing 6e -19
10 GW316489 Ribosomal rRNA Translation 1e-13
11 GW342893 Peltandra virginica- tRNA-Lys
(trnK) gene
Translation 2e-14
12 GW276092 tRNA Leu trnL - trnF IGS Translation 9e 75
13 GW342886 Beta macrocarpa-
mitochondrial genome
DNA synthesis/transcription and
Translation
1e-06
14 GW316482 A. thaliana- sec 61 beta-
subunit
Protein translocation to ER 3e-09
15 GW342894 GABA permease Translocation of GABA 8e-10
16 GW276089 O.sativa- E3 ubiquitin ligase UV B light response/low
temperature tolerance
9e-48
17 GW343027 P. trichocarpa- shikimate
kinase
Cold stress 9e-07
18 GW316730 Candida albicans- choline
kinase
Salinity stress 0.070
19 GW276087 Levan sucrase Bacillus subtilis Osmotic stress 2e-74
20 GW314859 Chlamydia trachomatis-
Phosphoglucoisomerase gene
Involved in synthesis of
galactomanan (osmoprotectant)
6e-43
21 GW276091 Talaromyces stipitatus- Sugar
transporter protein
Transport of sugar across the
membranes
1e- 24
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Sl.
No
Accession
Number Predicted gene family Putative Function
E-
value
22 GW343023 Cucumis melo- catalase 2 Oxidative stress 6e-08
23 GW342891 A. thaliana- Lipid associated
protein (fibrillin like proteins)
Cold/photooxidative stress 0.003
24 GW342892 Neosartorya fischeri-Integral
membrane protein
Cell structure maintenance 1e - 05
25 GW314854 A. thaliana- 60 α chaperonin sub
unit CCT family
Cold tolerance 6e-43
26 GW316481 Ricinus communis- ATP
binding protein
Osmotic stress 1e-22
27 GW342889 Cyanate hydratase Nitrogen metabolism 1e-12
28 GW316732 E.coli- serine deaminase
activator gene
Aminoacid metabolism 2e-21
29 GW316733 E.coli- BioH gene Lipid metabolism 6e-11
30 GW316485 S receptor kinases Self incompatibility 1e-18
31 GW276090 Populus sp- Drought stress
related protein
Drought stress tolerance 1e-38
32 GW314853 Populus trichocarpa- stress
protein
Stress tolerance 0.002
33 GW342887 Hydra magnipapillata- Putative
signal tranduction
Signal transduction 0.014
34 GW315344 A. thaliana genome Unknown -
35 GW316487 B. rapa genomic DNA clone Unknown -
36 GW314858 Oryza sativa genomic DNA-
chromosome 4, BAC clone
Unknown -
37 GW314852 Vitis vinifera clone Unkown -
38 GW315342 Populus trichocarpa clone Unknown -
39 GW316483 Oryza sativa japonica BAC
clone
Unknown -
40 GW316728 Populus trichocarpa- predicted
protein, mRNA
Unknown -
41 GW314856 Unknown Unknown -
42 GW315345 Unknown Unknown -
43 GW314851 Unknown Unknown -
Table 2.9- The Cold Stress Responsive Genes Isolated from Carrot using SSH
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The G protein function as molecular switches to regulate numerous cellular
responses such as proliferation, differentiation, responding to external environmental
signals, etc. Rho-related GTPase in plants (Rop) play an important role in plant growth
and development by acting as a signalling protein and also confers abiotic stress tolerance in
Arabidopsis (Shin et al., 2009) and upon cold stress in rice (Hashimoto & Komatsu 2007).
The enhanced level of similar proteins was observed in carrot that may be playing same
role in signalling mechanism in response to low temperature treatment.
Regulation of transcription and translation plays an important role in stress
alleviation (Miranda et al., 2003). RNA polymerase and maturase K are not only
involved in transcription and post-transcriptional modification, but have also been
implicated in regulation of gene expression through miRNA/siRNA formation in
response to stress in plants (Sunkar et al., 2007). There are reports of enhanced levels of
RNA polymerases and maturase K and a corresponding increase in the miRNA levels in
various plants exposed to biotic and abiotic stresses (Lee et al., 2005; Zhou et al., 2008;
Garavaglia et al., 2010).
Translation is regulated at the level of stability of transcripts and initiation of
translation (Prabu et al., 2011). Transcript stability under stress is enhanced by the formation
of polysomes (Arendt & Weidner 2011). In our study, carrot system upregulation of rRNA
was observed similarly that could have helped in the maintaining the transcript stability.
Translation initiation factor 4α also showed upregulation on exposure to cold stress and
similar results were also observed in plants like pea (Pham et al., 2000; Vashisht et al., 2005),
wheat (Kamal et al., 2010). Sec-β 61, which was upregulated in cold stressed carrot,
showed a similar response to wounding stress in Arabidopsis (Pnueli et al., 2003) and it
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is also essential for translocation of proteins to ER and for the cell viability as reported in
many of the organisms (Leroux & Rokeach 2008). In carrot, Sec β 61 might be
responsible for the translocation of different cold tolerant proteins to ER for processing.
Apart from that GABA permeases that translocate GABA to combat stresses, which was
also enhanced in the cold stressed carrot. GABA, a non-protein amino acid has been found to
increase in response to various stresses (Cholewa et al., 1997; Serraj et al., 1998; Bouche &
Fromm 2004).
Ubiquitin ligases are the group, whose expression was enhanced during the cold
stress in carrot. Similar E3 ubiquitin ligases (SIZ1) resulted in the accumulation of SUMO
protein conjugates that provided cold tolerance in Arabidopsis (Chinnusamy et al., 2007).
Sumoylation is a post-translational modification that protects the target protein from
proteosomal degradation by preventing ubiqutination.
Shikimate, derived compounds have a major role in plant response to biotic and
abiotic stresses (Hamberger et al., 2006), mainly due to shikimate kinase, the first
enzyme in the pathway is reported to play a key role in providing stress tolerance to
Arabidopsis (Fucile et al., 2008) and maize (Zheng et al., 2006). Thus, the upregulation
of shikimate kinase may probably increase the synthesis of metabolites, which confer
tolerance to cold stress in carrot. Cold stress has been reported to mimic water deficit
conditions similar to salinity stress (Mahajan & Tuteja 2005). Multiple mechanisms seem
to confer tolerance to the osmotic stress. Choline kinase catalyses the synthesis
of phosphatidylcholine responsible for maintaining the osmolority of the plant cell
(Tasseva et al., 2004) and upregulation of this gene in carrot may also have an important
role in cold tolerance.
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Cold stressed carrot showed upregulation of a transcript that had similarity with
the bacterial leavansucrase. Levansucrases are hexosyltransferases, mainly involved in
the metabolism of starch and sucrose and their expression in tobacco have been reported
to enhance the plant‘s osmotic tolerance (Park et al., 1999). Hence, we presume that the
carrot levansucrase might also be playing a similar role during low temperature stress.
Phosphoglucoisomerase catalyse the biosynthesis of galactomannans in plants
(Lee et al., 1984), which apart from their primary role as storage reserves in endosperm,
also provide osmoprotection during seed germination in response to external factors like
drought and low temperature in leguminous seeds (Mulimani & Prashanth 2002). Low
molecular weight sugars play a role as cryoprotectants as well as help to maintain
osmotic potential in different plant tissues. Sugar transporter proteins are involved in
distribution of sugars to various cells and tissues (Williams et al., 2000). In Arabidopsis,
ERD6, a putative sugar transporter protein (Kiyosue 1998) and tonoplast monosaccaharide
transporter proteins (Eckardt 2006) expressed in response to dehydration stress and during
general stress respectively.
The low temperature stress induced the generation of reactive oxygen species
(ROS), which have strong adverse effect on biomolecules and plant cell membranes
(Chaitanya et al., 2001). In plants, ROS are mitigated by the antioxidant enzymes like
catalase, peroxidase, and superoxide dismutase (Yong et al., 2008; Mallik et al., 2011).
Hence, these parameters were studied in cold stressed carrot, which showed that only
catalase is upregulated, which could be for combating the ROS generation. The prominent
role of catalase during low temperature stress has been already reported in wheat
(Apostolova et al., 2008) and maize (Prasad 1997). Fibrillins are a group of lipid binding
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proteins in plastids, which are induced during various types of abiotic stress responses,
like a combination of photooxidative and cold stress in Arabidopsis (Youssef et al., 2010),
low temperature stress in rice (Lee et al., 2007). In carrot, upregulation of fibrillins may
confer the stability of the plastids upon cold treatment.
Maintenance of membrane stability is important for stress tolerance (Gulen et al., 2008).
The low temperature stress affects the normal functioning of integral membrane proteins
and the activity of these proteins mainly depends on the fluidity of the cell membranes.
Xv SAP1 an integral protein, isolated from Xerophyta viscosa show high homology to
WCOR413, a cold responsive protein from wheat (Garwe et al., 2003). This shows that
the integral proteins are not only involved in maintaining the cell structure, they are also
associated with low temperature stress.
Chaperonin 60 and other kind of chaperonins are chloroplast proteins, thought to
play a key role in assembly and folding of proteins. As in the case of cold stressed carrot,
it has been reported to be cold induced in yeast (Somer et al., 2002), high temperature
stress in rice (Han et al., 2009). In cold stressed carrots the upregulation of chaperonins
and other chaperons could help in the correct folding of proteins expressed during the
cold stress. The proper folding of the membrane proteins would be a paramount
importance for the cold tolerance function. ATP binding proteins are group of membrane
proteins, involved in the transport of solutes across the cell membranes. Similar to the
results in carrot, they have been found to have a role in cold stress tolerance in rice
(Cui et al., 2005) as well as drought stress in Arabidopsis (Valliyodan & Nguyen 2006)
and for Ca dependent ATPases in response to cold stress in maize (Jian et al., 1999).
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The role of genes like cyanases, serine deaminases, S receptor kinase, etc. during
cold stress is not very clear yet. Though they have shown to have different roles in the
plant metabolic pathways, the upregulation might have direct/indirect role during the cold
stress, which has to be studied further. The comparative analysis of the transcriptome
showed that, most plants have a common mechanism to tolerate the low temperature
stress. From the current study, it is clear that the cold regulated transcriptome is
conserved in different plants species, though the level of up/down regulation vary among
different plant species.
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2.4 CONCLUSION
In this study, we have identified around 50 genes, which might play a role in
diverse function during the cold stress in carrot. Confirmation of the specific function of
each differentially expressed gene (from the forward library in carrot during cold
acclimation) will further require biochemical, molecular, physiological and genetic
analyses. Future studies on this aspect will help in increasing our understanding of the
complex mechanisms of abiotic stress response, in particular cold stress, which will
ultimately lead to the development of effective cold tolerant transgenic crops.
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