Newcastle University May 5, 2016 School of Biology Gene regulation under salt-stress; Differential alternative RNA splicing of the Δ 1 -Pyrroline-5- carboxylate Synthetase 1 (P5CS1) gene in Arabidopsis thaliana and Thellungiella salsuginea under salinity Mr Robert Fleming: 130211547 BIO3196: Biological Research Project Supervisor: Dr Tahar Taybi 2015/2016 1
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Newcastle University May 5, 2016
School of BiologyGene regulation under salt-stress; Differential alternative RNA splicing of the Δ1-Pyrroline-5-carboxylate Synthetase 1 (P5CS1) gene in Arabidopsis thaliana and Thellungiella salsuginea under salinity
Mr Robert Fleming: 130211547
BIO3196: Biological Research Project
Supervisor: Dr Tahar Taybi
2015/2016
1
Newcastle University May 5, 2016
Word Count: 8000
1. Abstract
Crop productivity is limited by environmental stresses including salt-stress. Proline accumulates in
leaves under stress conditions as an important osmoprotectant and anti-oxidant. The synthesis of
this important amino acid is controlled by the enzyme Delta1-pyrroline-5-carboxylate synthase
which is up regulated at the gene level by a variety of stresses. In this project intron-mediated
alternative RNA splicing as a means of regulating the P5CS1 gene was analysed under salt-stress
using RT-PCR technology in both the glycophyte, Arabidopsis thaliana and the halophyte,
Thellungiella salsuginea. Results confirmed P5CS1 to be induced by NaCl and showed a significant
difference in proline accumulation between the two plant species as well as between control
unstressed plants and plants subjected to salt-stress. In the leaves the splicing of some introns was
enhanced by salt-stress in Arabidopsis while in T. salsuginea splicing of the same introns was optimal
even in control plants. In roots however splicing of these introns was enhanced by salt-stress in both
species. Spatiotemporal regulation of the P5CS1 gene between plant organs is a likely explanation of
its control due to differential splicing in both the leaves and roots of plants when unstressed and
salt-stressed. The data shows differential regulation of the P5CS1 gene in glycophytes and
halophytes when subjected to salt-stress and highlights tissue specific regulation of the gene as a
possible factor contributing to salt-tolerance in halophytes. This provides promising applications in
biotechnology and agriculture when considering the optimisation of yields under stress but more
research is needed to ratify and apply the conclusions.
Key words: A. thaliana, T. salsuginea, salt, NaCl, salinity, stress, P5CS1, gene, regulation, differential,
intron, splicing, alternative, leaves, roots.
2
Newcastle University May 5, 2016
Contents
Abstract 2
Introduction 4
History and current developments in agricultural botany 4
Salt-stress as a significant abiotic stressor 4
Plant responses to salt-stress 5
Δ1-Pyrroline-5-carboxylate Synthetase 1 (P5CS1) gene and proline accumulation 5
Genome regulation as a factor conferring salt-tolerance 6
Aims and hypothesises 7
Methods 7
Materials and methods 8
Plant material and growth conditions 8
Proline determination and data analysis 8
gDNA extraction 9
Qualitative DNA PCR 9
RNA extraction 10
Qualitative RT-PCR 10
Agarose gel-electrophoresis 13
Results 13
Proline accumulation 13
Leaf gDNA and cDNA intron splicing 14
Root cDNA intron splicing 16
Discussion 18
Discussion of results 18
Limitations, critical appraisal and improvements to the study methods 20
Future work 22
Conclusion 24
Acknowledgements 25
3
Newcastle University May 5, 2016
References 25
Appendices 282. Introduction
2.1. History and current developments in agricultural botany
Agricultural botany underpins the development, evolution and ultimately the survival and
sustainability of mankind. It is the careful management and cultivation of crops that has driven and
formed the basis of today’s modern world. Science based agriculture became prevalent in the 20 th
century and significantly increased food production. Norman Borlaug, the father of the green
revolution, focused on breeding crop plants that increased the biomass they portioned to the grain
(Borlaug 2000). His work led to the development of lodging-resistant, high yielding, disease resistant
semi-dwarf grain varieties (Borlaug 2000). These varieties doubled crop yields in line with an
increasing demand for food and feed (Borlaug 2000). However, increasing yield through plant
breeding is somewhat exhausted and unsustainable. The semi-dwarf grain verities only did as well as
crop irrigation was becoming more sophisticated and farmers were applying more nutrients. Water
is a crucially limiting resource across the word, yet demand for it continues to soar. Additionally, the
most successful wheat plants invest approximately 60% of its resources into the grain (Borlaug
2000). It is unlikely that scientists can increase this any further. This highlights the importance of
identifying and developing novel methods to increase crop yields. Our planet is facing more evident
and pronounced challenges that were not as severe during the last green revolution and together
these factors further widen the gap between botanical sciences and the global food insecurity
phenomenon. To meet these demands and feed the increasing world population a 70% increase in
global food production is needed by 2050, which includes an additional 1 billion tonnes of cereal
crops (FAO 2009).
2.2. Salt-stress as a significant abiotic stressor
Sodium salts directly impact the survival of land plants. Our most valued terrestrial plants, the
cereals are classified as glycophytes and are particularly vulnerable to salt-stress as they die at salt
concentrations of approximately 100 mM NaCl (Munns and Tester 2008). Whereas, halophytic plants
such as, T. salsuginea (also T. halophila) can withstand NaCl concentrations of 500 mM (Wang et al.
2004). Nevertheless, biotechnology and agriculture are under ever increasing pressure as
approximately 1/5 of cultivated land is contaminated with salt, from which 1/3 of the worlds food
supply is produced and soil salinity is expected to result in 50% of arable land to be lost by 2050
(Wang et al. 2003). Due to this, extensive research has been carried out over the last 20 years to
4
Newcastle University May 5, 2016
understand mechanisms of stress-tolerance in order to develop crop plants that can survive in
extreme salt concentrations. This presents a possible field of scientific manipulation that can aid in
the alleviation of the global food insecurity challenge without crop land expansion.
2.3. Plant responses to salt-stress
Plant responses to salt-stress involve a downstream signalling cascade that aim at re-establishing
cellular osmotic pressure by maximising the production of osmoprotection proteins (Fleming 2015).
The outcome of the stress-signal perception, transduction and transcriptional up- or down-
regulation is the production of proteins and molecules with various plant protection, repair and
stabilisation functions, such as the osmoprotectant amino acid proline (Gong et al. 2005). These
mechanisms adjust the osmotic pressure back to optimal levels in order to maintain water uptake,
cell turgor and growth (Cabot et al. 2014). The ability of plants to respond to these stresses varies
greatly and are strongly linked to environmental selection pressures which have acted to enhance
the regulation of stress-response genes (Yeo et al. 1990). Science based agriculture now needs to
focus on identifying key genes that synthesise key proteins involved in stress-responses and
optimising their regulation in crop species. This will help science to produce crops that can survive
and grow in saline environments, helping to offset food insecurity.
2.4. Δ1-Pyrroline-5-carboxylate Synthetase 1 (P5CS1) gene and proline accumulation
P5CS1 is a stress-response gene with 20 introns in the model plant A. thaliana and 19 in its close
relative T. salsuginea. Alternative RNA splicing of the introns in the A. thaliana and T. salsuginea
P5CS1 gene are analysed in this report. P5CS1 encodes the enzyme delta1-pyrroline-5-carboxylate
synthase 1 (Hu et al. 1992). It catalyses the rate-limiting step of glutamate-derived proline
biosynthesis, increasing proline accumulation in response to salt-stress (Hu et al. 1992). This lowers
the water potential and subsequently induces expression of the gene throughout the whole plant
(Yoshiba et al. 1999), acting to trigger subcellular osmoregulatory stress-response pathways
(Strizhov et al. 1997). Proline is an essential compatible molecule and its production is part of a
common stress-response between A. thaliana and T. salsuginea (Gong et al. 2005). Transgenic
experiments have confirmed proline as a compatible osmolyte and a cryoprotectant but its
regulation and adaptive importance are yet to be fully concluded (Verbruggen and Hermans 2008).
Differential expression under salt-stress in A. thaliana and T. salsuginea have been shown to
correlate with higher P5CS1 transcript levels, higher levels of proline in the leaves and enhanced
5
Newcastle University May 5, 2016
control over Na+ uptake in T. salsuginea (Kant et al. 2006). This was further explored in the project.
However, further research is needed to confirm the factors regulating these responses.
2.5. Genome regulation as a factor conferring salt-tolerance
The ability of plants to respond optimally to salt-stress is vital to its long term survival in saline soils
and is notably different between A. thaliana and T. salsuginea (Vinocur and Altman 2005). It is now
widely recognised through extensive research into the mechanisms of salt-tolerance that differential
and spatiotemporal regulation of the expression of key stress-response genes, such as P5CS1 is
fundamental to salt-tolerance (Price et al. 2003). Metabolic plasticity is therefore crucial to plants’
survival in challenging environments. Understanding the mechanisms behind this plasticity in
halophytes is fundamental in order to provide the tools and knowledge of the regulation of salt-
tolerance for its applications in agriculture and biotechnology. This is because it determines the
rapidity of plants to mount a response to the stressor which significantly increases their resistance
and survival (Kesari et al. 2012). The halophytic and glycophytic regulation of the P5CS1 gene will be
considered throughout this report with a consideration of the possible practicalities of applying the
results obtained to C3 and C4 crops.
T. salsuginea has been showed to contain higher levels of proline when unstressed, and when
stressed it synthesises more proline than A. thaliana (Kant et al. 2006). Many hypothesises of the
salt-tolerance in T. salsuginea have been described. Firstly, the ortholog of the proline degradation
enzyme in A. thaliana (PDH) has been shown not to be expressed and is undetectable in the shoots
of T. salsuginea, indicating proline catabolism is strongly supressed (Kant et al. 2006). A higher basal
level of proline is thought to aid in the response T. salsuginea shows when exposed to salt-stress.
This is because it helps T. salsuginea mount an immediate and efficient response to the stressor.
Sequencing the genome of T. salsuginea also showed it to have a similar exon length to A. thaliana
but a far larger intron length of approximately 30% (Wu et al. 2012). This could also play a role in
determining gene expression regulatory functions such as, mRNA export and it may explain why T.
salsuginea has an enhanced control over its stress-response genes. The results obtained by Wu et al.
(2012) were further explored and built on in this project. These factors highlight the importance of
understanding the modulation of the transcriptome and proteome at the transcriptional and post-
transcriptional level under salt-stress conditions between A. thaliana and T. salsuginea. This is
6
Newcastle University May 5, 2016
because understanding the regulation of P5CS1 may aid in the elucidation of the mechanisms and
key regulators involved in the production of adequate physiological responses and their evolution in
different plant systems. The knowledge gained from this may be used in the production of crop
varieties with an enhanced tolerance to salt-stress that can be grown in previously inhabitable
environments.
2.6. Aims and hypothesises
This project aimed at observing and understanding the regulatory processes behind the differential
phenotypes of the glycophyte, A. thaliana and the halophyte, T. salsuginea when exposed to salt-
stress. The project aimed at answering the question as to whether the splicing of the P5CS1 gene is
induced by salt-stress and if there was a difference between A. thaliana and T. salsuginea? Focus
was on intron-mediated alternative mRNA splicing of the P5CS1 gene as a possible contributor to the
higher salt-tolerance shown by T. salsuginea comparatively to A. thaliana. Results show the
response to salt-stress at the tissue level between and within both species and provide some
preliminary data that begins to uncover halophytic and glycophytic regulation of the P5CS1 gene.
The project focused on qualitative observation of the splicing of the introns of the P5CS1 gene in A.
thaliana and T. salsuginea under control conditions and salt-stress. Secondly, through direct
observation to see if there was a difference between the splicing of the introns under control and
salt-stressed conditions between A. thaliana and T. salsuginea in both the leaves and roots. It was
hypothesised that T. salsuginea prepares its mature transcript significantly quicker than A. thaliana
in the leaves and roots and that intron-mediated splicing is working at full speed in both control and
salt-stressed conditions. This would mean that unlike A. thaliana, T. salsuginea mounts an
immediate response to salt-stress which confers its resistance to the abiotic stress.
2.7. Methods
Methods to obtain the results include: gDNA (leaves) and RNA (leaves and roots) extractions from
control and salt-stressed plants. The gDNA samples were extracted from the leaves of both plants
and were used to confirm the complete set of introns were present in both plant species when
exposed to control conditions (unstressed). Qualitative RT-PCR was performed on the RNA extracted
from both the water control plants and plants subjected to 100 mM NaCl for 3 days. This method
was used to reconvert the mRNA to cDNA from the water control and salt-stressed plants of both
species. Agarose gel-electrophoresis was used to run the samples in order to confirm the presence
7
Newcastle University May 5, 2016
of the introns of the P5CS1 gene in both plants in the gDNA controls of both species. It also enabled
the comparison of the splicing of introns in the coding region of P5CS1 in both the water control and
salt-stressed conditions between A. thaliana and T. salsuginea. This enabled a comparison to be
made between the mRNA splicing of the P5CS1 gene when exposed to control and salt-stressed
conditions in the leaves and roots both within and between species. Agarose gel-electrophoresis was
the best method to use as it allowed the experimenter to easily compare the response to salt-stress
between and within plant species and tissues.
3. Materials and methods
3.1. Plant material and growth conditions
A. thaliana (Columbia ecotype) and T. salsuginea (Shandong ecotype) seeds were surface sterilised
using 70% ethanol, washed three times with sterile water and sown on John Innes soil compost No.
3. The pots (12 cm wide) were placed at 4°C for 72 hours to synchronise germination. The pots were
then transferred to controlled growth room at 23°C with 12/12 hours light/dark periods and light
intensity of 150 μmol.m-2.s-1 at plant height. Seven-day-old seedlings were then transferred to
smaller pots (2.5 cm wide) containing moist John Innes No. 3 compost with one seedling in each.
Then 4-week-old A. thaliana and 6-week-old T. salsuginea plants, similar in size and before bolting,
were separated into three sets and irrigated with three different NaCl concentrations prepared with
normal tap water. A. thaliana was watered with 0, and 100 mM [NaCl] and T. salsuginea was
watered with 0, 100 [NaCl] (0 mM refers to tap water) at a fixed time (12:00) every day for 10 days.
Shoots and roots were harvested at a fixed time (16:00) as three plants per sample after 3 days of
the salt treatment, weighed and frozen in liquid nitrogen. Three samples were harvested at each
time point for each NaCl concentration for both plant species. Control plants were watered with tap
water only and harvested in parallel to salt-treated plants.
3.2. Proline determination
Nine plants in total were grown and leaf samples (second leaf from the shoot tip) from three 4-
week-old A. thaliana and three 6-week-old T. salsuginea plants were collected at 12 p.m. from the
water controls and plants subjected to 100 mM NaCl for 3 days. The extraction method and
colorimetric determination using acidic ninhydrin reagent were carried out based on previously
successful methods (Bates et al. 1973) but optimised to the specifics of this experiment. Volumes
and masses of ninhydrin were based on those used by Claussen (2005): 2.5 g ninhydrin/100 ml
consisting of glacial acetic acid, sterile water and 85% ortho-phosphoric acid in proportions of 6:3:1
8
Newcastle University May 5, 2016
(Claussen 2005). 10 ml of 3% (w/v) aqueous sulfosalicylic acid and quartz sand was added to a
mortar and 1 g of each leaf (FW) taken from each plant was ground using a pestle. Two layers of
glass-fibre filter (Schleicher & Schüll, GF 6, Germany) was then used to filter the homogenate. The
remains were discarded and the clear filtrate was used in the proline assay. 1 ml of ninhydrin and
glacial acetic were added to 1 ml of the filtrate. These were then transferred to a water bath set to
100°C for 1 hour. The reaction was terminated by transferring the reaction mixtures to a water bath
set to 21°C for 5 minutes. Colorimetric readings were recorded instantly at a wavelength of 546 nm.
The concentration of proline was determined from a standard curve using pure proline to quantify
the samples and calculated based on the μmol of proline per g of leaf fresh weight
(μmol proline (g FW)−1) (Claussen 2005).
3.2.1. Data analysis
There was no significant deviation between the variances of the residuals and normal distribution for
both A. thaliana and T. salsuginea. Therefore, a general linear model was used to model the effects
of plant species and salt-stress on proline accumulation.
3.3. gDNA extraction
Using the the Invisorb Spin plant Mini Kit II (Invitek, Germany) gDNA was extracted from both plant
species. Plant material was ground to a fine powder using liquid nitrogen. 400 µl of lysis buffer was
added to a 1.5 ml tube and 100 mg of ground plant tissue was added to this. 5 µl of proteinase K was
added to the 1.5 ml tube and then vortexed and incubated at 65°C for 30 minutes. The lysate was
transferred to a spin filter and spun at 12000 rpm in a mini-centrifuge for 1 minute at room
temperature. 200 µl of the binding buffer was added to the filtrate before being vortexed and then
the filtrate was placed on another spin filter and spun in the same conditions as before. The filtrate
was discarded and placed on a spin filter on a receiver tube and added to it was 550 µl of wash
buffer I before being spun again in the same conditions. This step was repeated again but this time
with 550 µl of wash buffer II. The filtrate was discarded and the spin filter was placed on a receiver
tube and spun in the same conditions again but this time to dry out the resin in the spin filter. The
product was then placed in a 1.5 ml tube and added to it 100 µl of the elution buffer (pre-warmed to
55°C). This was left to stand for 2 minutes at room before being spun in the same conditions to elute
the gDNA.
3.4. Qualitative DNA PCR
The following reagents were added to PCR tubes to make a 25 µl reaction: 1 µl gDNA (Table 3) or
cDNA, 1 µl of the forward primer (10 µM), 1 µl of the reverse primer (10 µM), 12.5 µl x2 MyFI Mix
9
Newcastle University May 5, 2016
(Bioline, UK) and 9.5 µl DEPC-water. PCR procedure was as follows: initialisation at 95°C for 5
minutes, the cyclical reactions ran for 35 cycles starting with a denaturation temperature of 94°C for
15 seconds, the annealing temperature was optimised to 58°C for 30 seconds and the extension
temperature was 72°C for 1 minute. Final extension was at 72°C for 5 minutes, final hold was set to
4°C until samples were removed. The lid temperature was set to 105°C. Samples were either used
immediately or stored at -20°C.
3.5. RNA extraction
Following the TRI-REAGENT method, plant material was ground to a fine powder using liquid
nitrogen and then in the fume hood, 1 ml Tri-reagent (Helena Biosciences, UK) was added to a 2 ml
RNase/DNase free tube. 150 mg of plant material was added and left to stand for 2 minutes before
shaking and inverting to mix the samples. The tube was then left to stand for 10 minutes at room
temperature. With care, 250 µl of chloroform was added, mixed, left at room temperature for 5
minutes and then spun at 13000 rpm at 4°C for 10 minutes. The upper phase was then transferred to
a 1.5 ml RNase/DNase free tube. 250 µl of 0.8 M Na citrate/1.2 M NaCl solution and 250 µl of
isopropanol was added. The solution was mixed and then then spun at 13000 rpm at 4°C for 30
minutes. The supernatant was then removed and the pellet washed with 1 ml of 70% ethanol,
vortexed and then spun at 13000 rpm at 4°C for 5 minutes. The supernatant was removed again and
the RNA pellet was left to air dry in the fume hood, taking care not to over dry the pellet. The RNA
pellet was then re-suspended in 20 µl of DEPC-water, vortexed and left on ice for 1 hour.
Concentration of RNA samples were read spectrophotometrically at 260/280 nm on the NanoDrop
Lite (Thermo Scientific, UK) and displayed in Table 4 and 5. RNA was extracted from 3 different
plants and mixed together for each condition and DNase treated before the RT-PCR.
3.6. Qualitative RT-PCR
Using the Tetro cDNA Synthesis Kit (Bioline, UK) RNA was reverse transcribed to cDNA. RNA samples
were first incubated at 65°C for 10 minutes and then put on ice for 2 minutes to open the RNA
molecules. All solutions were briefly vortexed and centrifuged before use. The priming mix was
prepared in an RNase-free PCR tube as follows: 5 µl of RNA per sample was added and the rest
frozen at -80°C for long term storage. 1 µl of the oligo (dT)18 primer, 10 mM dNTP mix, RiboSafe
RNase inhibitor and the Tetro Reverse Transcriptase (200 u µl-1) was then added to the same tube. 4
µl of the 5x RT buffer was added and finally 7 µl of DEPC-water was added to bring the total volume
10
Newcastle University May 5, 2016
to 20 µl. Samples were then mixed slightly by pipetting. RT-PCR reactions were as follows: samples
were incubated at 45°C for 30 minutes and then the reaction was terminated at 85°C for 5 minutes.
PCR reactions were carried out as described in 2.4. and the remaining cDNA was stored at -20°C for
long term storage.
Intron Primer Sequence Amplicon size (bp)Forward Reverse Unspliced Spliced
1
2
3
4
5
6 & 7
8
9
10
11
12
13
14
15
16
17
18
19
5’ – TCG TTA AGG TTC GTT GAG – 3’5’ – GAT TGG CTC TTG GTC GCT TA – 3’5’ – CTT GCG GAA TTA AAC TCG GAT G – 3’5’ – AAG CCT CAG AGT GAA CTT GAT G – 3’5’ – CTC AAC TTC TGG TGA ATG ACA G – 3’ 5’ – CCT AAC TCA AAG TTG ATC CAC AC – 3’5’ – ATA GAT AAA GTC CTC CGA GGA C – 3’5’ – TAT AAT ATC GCC GAC GCT CTT G – 3’5’ – AGT TCG TAA GCT AGC CGA TAT G – 3’5’ – AGT TCG TAA GCT AGC TGA TAT GG – 3’5’ – ACA GAT AGC TTC ACT TGC CAT C – 3’5’ – TGC CAT CCG TAG TGG AAA TG – 3’5’ – ATC ACT GAT GCA ATT CCA GAG A – 3’5’ – GCA ACA AGC TTG TTA CT – 3'5’ – GGA AAC TCT TCT TGT GCA TAA GG – 3’5’ – TCA CTG TAT ATG GTG GAC CAA G – 3’5’ – CAC ACA GAT TGC ATT GTG ACA G – 3’ 5’ – TTT TCC ACA ACG CAA GCA
5’ – ACG ACC AAG AGC CAA TCT TC – 3’5’ – GAC TAA TTG TCT GTA TCG AAG C – 3’5’ – CGA ACA TAG TCT CGT AAT AAG CC – 3’ 5’ – CTC TTC TGG TGC TTA TAG CAT C – 3’5’ – GTG TGG ATC AAC TTT GAG TTA GG – 3’5’ – GTG AAA GTT CCT AGA AAG CTT AG – 3’5’ – AAG AGC GTC GGC GAT ATT ATA C – 3’5’ – AAA ACA CGG CCA ATT GGA TCT TC – 3’5’ – CAT CAG GTC GGG ATT CAA AAA C – 3’5’ – GAC CAT CTG CCA CCT CTA AA – 3’5’ – GAG CAA ATC AGG AAT CTC TTC TC – 3’5’ – GAA GTC ACA AGT CCA ATG AGT TTA C – 3’5’ – GTT GCT TCC TCT TGG GAT CA – 3’ 5’ – CAT TAC AGG CTG CTG GAT AGT – 3’5’ – AAG CCT TGG AAC AGT ACT CAT AG – 3’5’ – GAA GGA ATA GCT CTG CAA CTT C – 3’5’ – CCA TCT GAG AAT CTT GTG CTT G – 3’5’ – GTA AGT AAT CCT TCA
347
281
223
318
396
351
288
347
270
204
257
217
211
244
150
302
200
179
59
134
107
238
293
250
191
229
137
71
171
135
126
159
69
204
115
87
11
Newcastle University May 5, 2016
20CAA G – 3’5’ – GTC GGA GTT GAA GGA TTA CTT AC – 3’
ACT CCG AC – 3’ 5’ – TCC TCA AGT CTC AAC ACA CAA C – 3’
179 76
Intron Primer Sequence Amplicon size (bp)Forward Reverse Unspliced Spliced
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
5’ – CTT CCC TCA CCA GAT ATT TCC – 3’5’ – ATT GGC TCT TGG TCG CCT AG – 3’5’ – CTT GCG GAA TTA AAC TCG GAT G – 3’5’ – AAG CCT CAG AGT GAA CTT GAT G – 3’5’ – CTC AAC TTC TGG TGA ATG ACA G – 3’ 5’ – CCT AAC TCA AAG TTG ATC CAC AC – 3’5’ – ATA GAT AAA GTC CTC CGA GGA C – 3’5’ – TAT AAT ATC GCC GAC GCT CTT G – 3’5’ – AGT TCG TAA GCT AGC CGA TAT G – 3’5’ – AGT TCG TAA GCT AGC TGA TAT GG – 3’5’ – ACA GAT AGC TTC ACT TGC CAT C – 3’5’ – TGC CAT CCG TAG TGG AAA TG – 3’5’ – ATC ACT GAT GCA ATT CCA GAG A – 3’5’ – GCA ACA AGC TTG TTA CT – 3'5’ – GGA AAC TCT TCT TGT GCA TAA GG – 3’5’ – TCA CTG TAT ATG GTG GAC CAA G – 3’5’ – CAC ACA GAT TGC ATT GTG ACA G – 3’
5’ – AGT GCT CCT AAG CGA CCA AG – 3’5’ – TCT GTA TCG AAG CCT TTG CC – 3’5’ – CGA ACA TAG TCT CGT AAT AAG CC – 3’ 5’ – CTC TTC TGG TGC TTA TAG CAT C – 3’5’ – GTG TGG ATC AAC TTT GAG TTA GG – 3’5’ – GTG AAA GTT CCT AGA AAG CTT AG – 3’5’ – AAG AGC GTC GGC GAT ATT ATA C – 3’5’ – AAA ACA CGG CCA ATT GGA TCT TC – 3’5’ – CAT CAG GTC GGG ATT CAA AAA C – 3’5’ – GAC CAT CTG CCA CCT CTA AA – 3’5’ – GAG CAA ATC AGG AAT CTC TTC TC – 3’5’ – GAA GTC ACA AGT CCA ATG AGT TTA C – 3’5’ – GTT GCT TCC TCT TGG GAT CA – 3’ 5’ – CAT TAC AGG CTG CTG GAT AGT – 3’5’ – AAG CCT TGG AAC AGT ACT CAT AG – 3’5’ – GAA GGA ATA GCT CTG CAA CTT C – 3’5’ – CCA TCT GAG AAT CTT GTG CTT G – 3’
723
278
374
352
404
503
290
300
250
184
275
235
211
155
287
303
190
216
124
202
238
293
306
187
197
137
71
171
135
126
73
162
204
107
12
Table 2. The sequences of each primer base pair and predicted amplicon size for both unspliced and spliced introns of the
P5CS1 coding sequence in Thellungiella salsuginea. Amplicon sizes (bp) were calculated for introns 1-19. Primers from
Integrated DNA Technologies, Belgium.
Table 1. The sequences of each primer base pair and predicted amplicon size for both unspliced and spliced introns of the
P5CS1 coding sequence in Arabidopsis thaliana. Amplicon sizes (bp) were calculated for introns 1-20. Introns 6 and 7
were amplified as a single amplicon. Primers from Integrated DNA Technologies, Belgium.
0.1292 μmol (g FW)−1 in T. salsuginea. Proline accumulation also increased by 0.1208 μmol (g FW)−1
in T. salsuginea when exposed to 100 mM of NaCl for 3 days. This suggests that plant species has a
slightly stronger influence on proline accumulation than salt-stress (when measured in μmol (g FW)−1) although both factors have shown to effect proline accumulation similarly. Figure 1 shows that
proline concentration in both A. thaliana and T. salsuginea is greater when stressed than when
unstressed. T. salsuginea has a higher basal level of proline than A. thaliana when unstressed and
higher levels again when stressed (Figure 1). Additionally, Figure 1 shows that when unstressed, T.
salsuginea accumulates almost the same concentration of proline as A. thaliana does when salt-
Claussen W (2005) Proline as a measure of stress in tomato plants. Plant Science 168:241-248.
FAO UN How to Feed the World in 2050. In: Rome: High-Level Expert Forum, 2009.
Fleming R (2015) Regulation of P5CS1 gene, determining the mechanisms of Salt tolerance as a
possible contributing solution to growing food insecurity. Dissertation, Newcastle University
Ghars MA, Parre E, Debez A, Bordenave M, Richard L, Leport L, Bouchereau A, Savouré A, Abdelly C
(2008) Comparative salt tolerance analysis between Arabidopsis thaliana and Thellungiella
halophila, with special emphasis on K+/Na+ selectivity and proline accumulation. Journal of
plant physiology 165:588-599.
Gong Q, Li P, Ma S, Indu Rupassara S, Bohnert HJ (2005) Salinity stress adaptation competence in the
extremophile Thellungiella halophila in comparison with its relative Arabidopsis thaliana.
The Plant Journal 44:826-839.
25
Newcastle University May 5, 2016
Holst-Jensen A, Rønning SB, Løvseth A, Berdal KG (2003) PCR technology for screening and
quantification of genetically modified organisms (GMOs). Analytical and Bioanalytical
Chemistry 375:985-993.
Hu CA, Delauney AJ, Verma DP (1992) A bifunctional enzyme (delta 1-pyrroline-5-carboxylate
synthetase) catalyzes the first two steps in proline biosynthesis in plants. Proceedings of the
National Academy of Sciences 89:9354-9358.
Iida K, Seki M, Sakurai T, Satou M, Akiyama K, Toyoda T, Konagaya A, Shinozaki K (2004) Genome-
wide analysis of alternative pre-mRNA splicing in Arabidopsis thaliana based on full-length
cDNA sequences. Nucleic acids research 32:5096-5103.
Kant S, Kant P, Raveh E, Barak S (2006) Evidence that differential gene expression between the
halophyte, Thellungiella halophila, and Arabidopsis thaliana is responsible for higher levels
of the compatible osmolyte proline and tight control of Na+ uptake in T. halophila. Plant, Cell
& Environment 29:1220-1234.
Kesari R, Lasky JR, Villamor JG, Des Marais DL, Chen Y-JC, Liu T-W, Lin W, Juenger TE, Verslues PE
(2012) Intron-mediated alternative splicing of Arabidopsis P5CS1 and its association with
natural variation in proline and climate adaptation. Proceedings of the National Academy of
Sciences 109:9197-9202.
Mattioli R, Falasca G, Sabatini S, Altamura MM, Costantino P, Trovato M (2009) The proline
biosynthetic genes P5CS1 and P5CS2 play overlapping roles in Arabidopsis flower transition
but not in embryo development. Physiologia Plantarum 137:72-85.
Munns R (2002) Comparative physiology of salt and water stress. Plant, Cell & Environment 25:239-
250.
Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annual Review of Plant Biology 59:651-
681.
Nishimura N, Kitahata N, Seki M, Narusaka Y, Narusaka M, Kuromori T, Asami T, Shinozaki K,
Hirayama T (2005) Analysis of ABA hypersensitive germination2 revealed the pivotal
functions of PARN in stress response in Arabidopsis. The Plant Journal 44:972-984.
Price TD, Qvarnström A, Irwin DE (2003) The role of phenotypic plasticity in driving genetic evolution.
Proceedings of the Royal Society of London B: Biological Sciences 270:1433-1440.
Strizhov N, Abraham E, Ökrész L, Blickling S, Zilberstein A, Schell J, Koncz C, Szabados L (1997)
Differential expression of two P5CS genes controlling proline accumulation during salt stress ‐requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis. The Plant Journal
12:557-569.
Verbruggen N, Hermans C (2008) Proline accumulation in plants: a review. Amino acids 35:753-759.
26
Newcastle University May 5, 2016
Vinocur B, Altman A (2005) Recent advances in engineering plant tolerance to abiotic stress:
achievements and limitations. Current opinion in biotechnology 16:123-132.
Wang W, Vinocur B, Altman A (2003) Plant responses to drought, salinity and extreme temperatures:
towards genetic engineering for stress tolerance. Planta 218:1-14.
Wang Z-l, Li P-h, Fredricksen M, Gong Z-z, Kim CS, Zhang C, Bohnert HJ, Zhu J-K, Bressan RA,
Hasegawa PM (2004) Expressed sequence tags from Thellungiella halophila, a new model to
study plant salt-tolerance. Plant Science 166:609-616.
Wu H-J, Zhang Z, Wang J-Y, Oh D-H, Dassanayake M, Liu B, Huang Q, Sun H-X, Xia R, Wu Y (2012)
Insights into salt tolerance from the genome of Thellungiella salsuginea. Proceedings of the
National Academy of Sciences 109:12219-12224.
Yeo AR, Yeo ME, Flowers SA, Flowers TJ (1990) Screening of rice (Oryza sativa L.) genotypes for
physiological characters contributing to salinity resistance, and their relationship to overall
performance. Theoretical and Applied Genetics 79:377-384.
Yoshiba Y, Nanjo T, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Stress-responsive and
developmental regulation of Δ 1-pyrroline-5-carboxylate synthetase 1 (P5CS1) gene
expression in Arabidopsis thaliana. Biochemical and biophysical research communications
261:766-772.
Yu S, Wang W, Wang B (2012) Recent progress of salinity tolerance research in plants. Russian
Journal of Genetics 48:497-505.
8. Appendices
27
Table 3. gDNA concentrations (ng µl-1) of extracts from 4-week-old A. thaliana and 6-week-old T. salsuginea
control plants. gDNA extracted from the leaves and used in the PCR. gDNA samples were read
spectrophotometrically at 260/280 nm on the NanoDrop Lite (Thermo Scientific, UK). A260/A280 values
greater than 1.8 are suitable for analysis.
Plant Species gDNA concentration (ng µl-1) A260/A280
A. thalianaT. salsuginea
28.00285.5
2.041.86
Newcastle University May 5, 2016
Plant conditionsA260/A280 Leaves A260/A280 Roots
A. thaliana T. salsuginea A. thaliana T. salsuginea
Water control100 mM Nacl for 3 days
2.132.14
2.142.17
2.092.15
2.192.17
(A) Arabidopsis thaliana P5CS1 gene sequence taken from the NCBI database. Highlighted in pink is 5’ flanking sequence, in yellow are start and stop codons of the CDS, in aqua blue are the exons, in grey are the introns and in red is the 3’ flanking sequences.
(B) Thellungiella salsuginea P5CS1 gene taken sequence from the Phytozorm database (unpublished). Highlighted in pink is 5’ flanking sequence, in yellow are the start and stop of the CDS, in aqua blue are the exons, in grey are the introns and in red is the 3’ flanking sequences.