Dissertation thesis

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


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


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

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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


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


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


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


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


(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


(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


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


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.


18

19

5’ – TTT TCC ACA ACG CAA GCA CAA G – 3’5’ – GTC GGA GTT GAA GGA TTA CTT AC – 3’

5’ – GTA AGT AAT CCT TCA ACT CCG AC – 3’ 5’ – TCC TCA AGT CTC AAC ACA CAA C – 3’

213

273

121

142

3.7. Agarose gel-electrophoresis

1.5% agarose gels were made by weighing 3 g of agarose (Molecular grade) and put in a conical flask.

200 ml of x0.5 tris boric acid EDTA (TBE) was added to this and then the contents swirled to mix

them. The agarose was then melted in in micro-waive and once melted it was left to cool. Once

cooled and when wearing gloves and goggles 9 µl of Ethidium Bromide (stock solution) was added

and the conical flask swirled. The agarose solution was then poured into a pre-prepared gel tray and

left to solidify for 30 minutes. The gel was then placed in a gel box and submerged in x0.5 TBE. 2 µl

of the 100 base pair molecular size marker (Bioline, UK) was loaded as well as 5 µl of g/cDNA with 2

µl of the x6 loading dye (Bioline, UK) for each of the 19/20 introns studied. Samples were run for 1

hour at 100 V and then gels were visualised under UV light using a gel-doc system.

4. Results

4.1. Proline accumulation

Both plant species and salt-stress (Figure 1) had a significant effect on proline accumulation (ANOVA,

Plant species: F1,9 = 18.95, p = 0.002; salt-stressed: F1,9 = 16.59, p = 0.003). R2 = 79.79% of variation in

the proline concentration was explained by the plant species and the NaCl concentration. The

13

A. thaliana T. salsuginea 0

0.1

0.2

0.3

0.4

0.5

0.63 days water 3 days 100 mM NaCl

Plant Species

Prol

ine

Conc

entr

ation

(μm

ol (g

FW

)−1)

± s.

e.

Figure 1. Proline concentration (μmol proline (g FW)−1) in Arabidopsis thaliana and Thellungiella

salsuginea subjected to control (3 days of water) and salt-stress (3 days of 100 mM NaCl)

conditions. 9 plants in total were grown and leaves were taken at midday from 3 of the plants and

ground together (n = 3,3,3,3). Error bars are ± 1 standard error.


regression equation was proline concentration = 0.2158 - 0.1292 plant_A. thaliana + 0.1292 plant_T.

salsuginea - 0.1208 H2O / NaCl_H2O + 0.1208 H2O / NaCl_NaCl. Proline accumulation increased by

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-

stressed.

4.2. Leaf gDNA and cDNA intron splicing

14

M 1 2 3 4 5 6 & 7 8 9 10 11 12 13 14 15 16 17 18 19 20

A

CFigure 2. Agarose gels of Introns 1-20 in the leaves of the Arabidopsis thaliana P5CS1 gene using Ethidium

bromide (stock solution) to stain the gel, x0.5 TBE buffer, x6 loading dye (Bioline, UK) and M = the 100

base pair molecular size marker (Bioline, UK). A = gDNA control, B = cDNA water control and C = cDNA

after 3 days of 100 mM NaCl. Gels viewed under UV light using a gel-doc system. gDNA and RNA isolated

from the leaves.

1000 bp

500 bp400 bp300 bp200 bp100 bp

1000 bp

500 bp400 bp300 bp200 bp100 bp

1000 bp

500 bp400 bp300 bp200 bp100 bp

1000 bp

500 bp400 bp300 bp200 bp100 bp

1000 bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

V

B


Figure 2 displays the intron splicing pattern between the controls and salt-stressed A. thaliana and

Figure 3 displays the pattern in T. salsuginea with predicted spliced and unspliced transcript lengths

per intron shown in Table 1 and 2. Both Figure 2 and 3 show that intron splicing and preparation of

mRNA is different within and between both plant species under control and salt-stressed conditions.

The gDNA control shown in image A of Figure 2 and image V of Figure 3 verifies there was no

contamination in the samples. It also confirmed that all 20 introns are present in the A. thaliana

P5CS1 gene, all 19 introns are present in the T. salsuginea P5CS1 gene and that there is a clear

difference between the splicing of the introns between the gDNA of both plants. The gDNA controls

show that the PCR has been optimised to the most suitable conditions required for DNA

amplification and gives the experimenter confidence in subsequent PCR assays. This enabled the

successive analysis of intron splicing in both plant species under control and salt-stressed conditions

and for a comparison to be made of between the regulation of the P5CS1 splicing both between and

within the two plant species.

Gel image B and C of Figure 2 shows the amplicon sizes (Table 1) for all 20 introns in the mRNA of

the P5CS1 gene under unstressed (water control) and subsequently salt-stressed conditions in A.

thaliana. However, no amplicon for intron 2 of A. thaliana under salt-stressed conditions was

amplified. Therefore, no analysis of the intron splicing for intron 2 under salt-stress can be made

both within and between the species. When comparing this to image C of Figure 2, there is a clear

differential pattern of intron-mediated splicing of the P5CS1 gene. Figure 2 shows the greatest

difference in intron splicing between A. thaliana controls and salt-stressed plants was shown to be

between introns 4, 5, 6, 7, 8 and 9 as only under salt-stress was the splicing of those introns more

optimal and working at full speed (image B, C). Figure 2 also shows that under both unstressed and

stressed conditions (image B, C) the splicing of the introns in the P5CS1 gene in A. thaliana was never

working at full speed. Nevertheless, Figure 2 shows intron splicing was enhanced between water

control and salt-stressed A. thaliana (image B, C).

Gel image W and X of Figure 3 shows the amplicon sizes (Table 2) for all 19 introns in the mRNA of

the P5CS1 gene under unstressed (water control) and subsequently salt-stressed conditions in T.

salsuginea. However, no amplicon for intron 7 in of T. salsuginea under control and salt-stressed

conditions was amplified. Therefore, no analysis of the intron splicing under unstressed and salt-

stress conditions can be made both within and between species. When comparing image W to image

15

1000 bp

500 bp400 bp300 bp200 bp100 bp

1000 bp


X of Figure 3, there was no difference in the intron-mediated splicing of the P5CS1 gene in T.

salsuginea.

Halophytic and glycophytic differential intron-mediated alternative RNA splicing of the P5CS1 has

been has been shown under control and salt-stressed conditions in A. thaliana and T. salsuginea

(Figure 2, 3). Figure 2 shows A. thaliana prepares its mature transcripts of the P5CS1 gene quicker

under stress and splicing of introns 4, 5, 6, 7, 8 and 9 was particularly enhanced under salt-stress

(image B, C). However, Figure 3 shows that intron splicing in T. salsuginea was not enhanced under

salt-stress as splicing is already working at full speed in control plants (image W, X). Figure 2 (image

B) and Figure 3 (image W) shows that A. thaliana and T. salsuginea regulate the splicing of their

introns differently under unstressed conditions and that T. salsuginea has fewer unspliced mRNA

transcripts. Figure 2 (image C) and Figure 3 (image X) shows a different pattern of intron splicing

under salt-stress between both species and that T. salsuginea has fewer unspliced mRNA transcripts

than A. thaliana.

4.3. Root cDNA intron splicing

Introns 5, 6, 7, 8 and 9 were analysed in the roots as the optimal splicing and regulation of these

introns were thought to play an essential role in proline accumulation and salt-tolerance in P5CS1

genes expressed in the leaves. Additionally, root splicing was analysed as the P5CS1 gene is known

to be expressed in the roots but proline does not to accumulate there. This is because proline is

translocated to the leaves.

16

1000 bp

500 bp400 bp300 bp200 bp100 bp

1000 bp

Figure 4. Agarose gels of Introns 5, 6, 7, 8 and 9 in the roots of the

Arabidopsis thaliana P5CS1 gene using Ethidium bromide (stock

solution) to stain the gel, x0.5 TBE buffer, x6 loading dye (Bioline, UK)

and M = the 100 base pair molecular size marker (Bioline, UK). D =

cDNA water control and E = cDNA after 3 days 100 mM NaCl. Gels

viewed under UV light using a gel-doc system. RNA isolated from the

roots.

M 5 6 & 7 8 9 M 5 6 & 7 8 9 D E


Figure 4 displays the pattern of intron splicing of introns 5, 6, 7, 8 and 9 in the P5CS1 gene in

unstressed and salt-stressed conditions in A. thaliana and Figure 5 displays the splicing of introns 5,

6, 8 and 9 in T. salsuginea. Image D and E of Figure 4 shows that splicing was enhanced under salt-

stress in A. thaliana in a similar pattern to leaf splicing shown in Figure 2. Again, E shows that under

stressed conditions splicing of intron 6 and 7 was not working at full speed (Figure 4). This agrees

with the data shown in image C of Figure 2 and shows that in the roots and leaves splicing of intron 6

and 7 is not optimal and not working at full speed when A. thaliana was stressed. Image Y and Z of

Figure 5 also show the splicing of introns 5, 6, 8 and 9 in the T. salsuginea P5CS1 gene to be

enhanced and working at full speed under salt-stressed conditions. Splicing of introns 5, 6, 8 and 9 in

the roots under salt-stress (Figure 5) produces a fragment with the predicted fragment length (Table

2). However, in the leaves, the length of intron 8 is approximately 100 bp larger (Figure 3) than that

of the roots in both control (spliced fragment), salt-stressed (Figure 5) and the expected fragment

length (Table 2). Additionally, intron splicing in the water control (image Y) is not optimal (Figure 5).

This is different to the results shown in image X of Figure 3, as splicing of introns 5, 6, 8 and 9 in the

leaves of the water control was optimal and working at full speed. Figure 4 and 5 (image D and Y)

show that splicing is more optimal and working at a faster speed in A. thaliana but when salt-

stressed (image E and Z) T. salsuginea has optimal splicing of all introns unlike A. thaliana.

5. Discussion

5.1. Discussion of results

17

1000 bp

500 bp400 bp300 bp200 bp100 bp

1000 bp

Figure 5. Agarose gels of Introns 5, 6, 8 and 9 in the roots of the

Thellungiella salsuginea P5CS1 gene using Ethidium bromide (stock

solution) to stain the gel, x0.5 TBE buffer, x6 loading dye (Bioline, UK)

and M = the 100 base pair molecular size marker (Bioline, UK). Y =

cDNA water control and Z = cDNA after 3 days 100 mM NaCl. Gels

viewed under UV light using a gel-doc system. RNA isolated from the

roots.

M 5 6 8 9 M 5 6 8 9 Y Z


The results have confirmed many of the aims and hypothesises and provide some preliminary data

on the regulation of the P5CS1 gene in glycophytes and halophytes. Research by eco-physiologists

and biochemists have shown that proline accumulation is greater in T. salsuginea in comparison to

A. thaliana and this is vital to its survival in saline soils (Ghars et al. 2008). The results displayed in

Figure 1 confirm this by clearly showing the extremophile T. salsuginea to accumulate more proline

under control conditions and salt-stress. This suggests that T. salsuginea constitutively expresses its

P5CS1 gene and that mechanisms are in place to inhibit proline catabolism. This could be due to T.

salsuginea not containing the proline degradation enzymes that A. thaliana does (Kant et al. 2006).

Therefore, there is substantial evidence showing that these factors enable T. salsuginea to mount an

efficient response to salt-stress and that this enables its survival in saline soils. However, it is also

important to look at the genome wide response to salt-stress. This is because many other genes,

such as PPC1 are known to be upregulated in response to salt-stress and 75 salt-responsive proteins,

such as glycine betane have been identified in T. salsuginea (Chang et al. 2015). This suggests that

the P5CS1 gene is part of an extensive, integrated and precisely managed molecular and

physiological response to salt-stress in T. salsuginea that still requires vast research to confirm the

mechanisms of stress-tolerance.

Figure 2 provides evidence suggesting that salt-stress induces the expression of the P5CS1 gene. This

is because intron splicing is enhanced when comparting the control A. thaliana plants to salt-

stressed (Figure 2). This means that the P5CS1 gene in A. thaliana is differentially regulated under

salt-stress. This suggests the gene is expressed under salt-stress conditions and mRNA is spliced

more quickly in order to prepare the mature transcripts at a faster rate. This is needed to respond

optimally to salt-stress. Therefore, posttranscriptional modification and regulation by intron-

mediated alternative splicing of these introns in A. thaliana and T. salsuginea is a significant factor

determining their responses to salt-stress (Figure 2, 3). However, there is regulation of the P5CS1

gene at every level but the preparation of mature transcripts is never fully optimal in A. thaliana

(Figure 2). This is because the splicing of introns 5, 6, 7, 8 and 9 have been shown not to be working

at full speed even after 3 days of salt-stress (Figure 2, image C). This proposes a factor that may

result in A. thaliana showing increased sensitivity to salt-stress in comparison to T. salsuginea.

The results also provide a possible explanation of why T. salsuginea has been shown to contain

higher concentrations of proline in both unstressed and salt-stressed plants (Figure 1). Under control

and salt-stressed conditions, the splicing of the introns in the P5CS1 gene in the leaves is different

between A. thaliana and T. salsuginea (Figure 2, 3). However, there is also no difference in intron

splicing of the gene when unstressed/salt-stressed in the leaves of T. salsuginea (Figure 3). This

suggests that T. salsuginea prepares its mature transcript extremely quickly and that RNA splicing is

18


working at full speed both when unstressed and salt-stressed. It also suggests that optimal splicing in

the halophyte may account for its ability to mount an immediate response to salt-stress which is

essential to its survival in saline soils. This is phenotypically shown by its survival in saline soils and

elevated proline levels (Figure 1). Comparing image C of Figure 2 and image X of Figure 3 shows leaf

intron splicing to be only fully optimal in T. salsuginea as opposed to A. thaliana. This provides

further evidence as to why T. salsuginea accumulates more proline than A. thaliana under control

and salt-stress (Figure 1). Intron 19 is unlikely to be important in salt-tolerance as in both control and

salt-stressed T. salsuginea show semi-optimal splicing (Figure 3).

Figure 4 and 5 show root expression and splicing of introns 5, 6, 7, 8 and 9 in the P5CS1 gene in A.

thaliana and introns 5, 6, 8 and 9 in T. salsuginea. Figure 4 and 5 show that salt-stress enhances

intron splicing in the roots of both plants. Image D of Figure 4 and image Y of Figure 5 suggest that in

the roots under control conditions splicing is working slightly faster in A. thaliana. This was not

expected as results from the leaves and previous research has shown splicing of P5CS1 gene to

always be optimal in T. salsguinea. However, Figure 4 (image E) and 5 (image Z) show that under

salt-stress intron splicing was only working at full speed in T. salsuginea. This was expected due to

previous studies on salt-tolerance in T. salsuginea and the results displayed in Figure 3. It can be

deduced from this that mRNA transcripts of the P5CS1 gene are prepared more quickly in both the

leaves and roots of T. salsuginea under salt-stress and that this is a vital to its tolerance to high

concentrations of NaCl. It is also worth noting the importance of spliced fragments when salt-

stressed, as optimal splicing under stress is vital to the resistance shown in T. salsuginea. This may

explain why the presence of unspliced fragments in the water control of the roots in T. salsuginea

does not greatly impact its ability to accumulate proline. Comparing Figure 2 and 4 shows that the

splicing of intron 6 and 7 in A. thaliana under both unstressed and salt-stressed conditions was never

working at full speed. This may be a significant factor inhibiting the production of mRNA transcripts

and may result in A. thaliana phenotypically showing a slower response to salt-stress.

The results for leaf and root P5CS1 intron splicing shows there is a difference in splicing both

between and within the organs of both plant species. The preliminary findings suggest

spatiotemporal regulation of the gene among different plant organs. Both leaf and root splicing

appears to be enhanced by salt-stress in A. thaliana and only root intron splicing is shown to be

enhanced in T. salsuginea subjected to salt-stress. Therefore, optimal splicing under salt-stress is a

likely component of an efficient response to the stressor. Only T. salsuginea shows splicing to be

working at full speed in both the leaves and the roots in control (leaves) and salt-stressed (leaves

and roots) conditions. This poses a new explanation for the salt-tolerant phenotype observed in T.

19


salsuginea. It also highlights the potential of optimising the regulation of stress-response genes in A.

thaliana and subsequently C3 and C4 crops.

5.2. Limitations, critical appraisal and improvements to the study methods

Potential limitations and criticality include the controversy over the units used to measure proline

accumulation (μmol proline (g FW)−1). This is because some molecular biologists may argue that salt-

stress may in turn cause water-stress and this would result in the experimenter taking a greater FW

of tissue from salt-stressed plants. However, it is now well known that salt-stressed plants recover

their water content after a brief period of osmotic unbalance (Munns 2002). This means that there

was no bias when taking 1 g (FW) of leaf samples from both control and salt-treated A. thaliana and

T. salsuginea. Therefore, no misrepresentational data of proline concentration in any of the plants

will have been reported.

Additionally, no amplicon for some introns was achieved and the time constraint of the project

meant the optimisation of the primers for those introns was not possible. A longer period of time (24

weeks) to collect the data would have allowed the experimenter to optimise all primers for all

introns. This would have meant that all introns could have been amplified and that the splicing of

these could have been compared both within and between species. This would have enabled the

analysis of the splicing for all 20 introns in A. thaliana and all 19 in T. salsuginea and may have

shown other introns that play a significant role in the salt-tolerance observed in T. salsuginea.

Introns 6 and 7 of the P5CS1 gene in A. thaliana was analysed and amplified together. This means it

was difficult to determine the splicing of both introns individually. Due to the close proximity of the

introns in the gene sequence designing individual primers for both introns was not possible.

Due to the time constraints of the project, only introns 5, 6, 7, 8 and 9 of the P5CS1 gene in A.

thaliana and only introns 5, 6, 8 and 9 in T. salsuginea were analysed in the roots under control and

salt-stressed conditions. Preliminary findings suggest spatiotemporal regulation of the gene is

different among plant organs and tissues in both plant species. Therefore, extending the period of

time allocated to study alternative splicing in both plants (24 weeks) would have enabled all introns

in the roots of both plants to have been analysed. This would resulted in greater knowledge of the

splicing of all introns in the roots of both plants under control and salt-stressed conditions and may

have provided further information on why T. salsuginea is a halophyte.

Additionally, a further improvement could have been to analyse the production of proteins from

unspliced transcripts. This could have shown whether the unspliced amplicons shown in Figure 2, 3,

4 and 5 were producing any proline biosynthesising enzymes. This would have assisted in the

20


confromation of proline accumulation and helped to understand and determine more accurately

how both plant species respond to salt-stress.

Due to the time constraints of the project, intron splicing was only analysed after 3 days of salt-

stress. It would have been better to look at splicing at days: 1, 3, 5, 7 and 10 in order to understand

at a greater level the pattern of intron splicing between A. thaliana and T. salsuginea under NaCl

concentrations of 100 mM. It is known that by day 10 the level of the P5CS1 transcript is the same in

A. thaliana and T. salsuginea but transcript levels plateau at day 3 in T. salsuginea. Investigating this

would aid in the understanding of why A. thaliana is slower at preparing its mature transcript.

Additionally, due to time restrictions only 100 mM of NaCl and its impact on intron splicing between

both plants was analysed. Treating A. thaliana and T. salsuginea to NaCl concentrations of 300 mM

and 500 mM as well as 100 mM would give a greater understanding of how increasing the

concentration of the stressor (NaCl) effects intron splicing and the preparation of mature P5CS1

transcripts in glycophytes and halophytes. Splicing could be analysed in a similar manner to other

studies (Iida et al. 2004) as well as the methods used in this study. Results would potentially show

how both plants respond to the initial onset of varying intensities of salt-stress and potentially aid in

confirming the characteristic rapid response shown in T. salsuginea.

5.3. Future work

There are still many areas that need to be investigated to give a complete and more rounded

knowledge of the regulation of the P5CS1 gene in both glycophytes and halophytes. Firstly, science

needs to determine what factors lead to the enhanced splicing and differential expression in T.

salsuginea? Is it the regulation by the splicesome or the differences in the intron sequences

between both species (Wu et al. 2012)? Further research should focus on investigating the role of

the splicesomes in the P5CS1 gene of A. thaliana and T. salsuginea as well as the differences in base

pair composition of the intron sequences between both species. This will help to uncover the role

the intron sequences and splicesomes play in salt-stress. This could then be applied to crop species

in the anticipation of improving the regulation of their stress-response genes in order to increase

crop yields. Engineering the P5CS1 gene of T. salsuginea into A. thaliana will help to answer these

questions. This is because it would show whether glycophytes have the capacity to regulate the

P5CS1 gene in the same way halophytes do. Therefore, if A. thaliana is unable to regulate the P5CS1

gene of T. salsuginea in the same way as T. salsuginea does, this would suggest that the splicesomes

21


are crucial to the enhanced splicing and production of mRNA seen in T. salsuginea. However, if A.

thaliana shows proline levels and splicing similar to that of T. salsuginea then this would suggest that

it is the differences in the intron sequences between A. thaliana and T. salsuginea that confer its

resistance to salt.

Further research should also focus on the differential impact of other stresses on the splicing of the

introns in both plants. This would show if other introns are differentially spliced under other abiotic

stresses such as, drought and heat stress. A comparison of intron splicing of salt, drought and heat

stress could then be made between and within unstressed and stressed A. thaliana and T.

salsuginea. This would show the imact of different abiotic stresses on intron splicing in both plants. It

may show splicing of certain introns to be more important to the stress-response of each abiotic

stress in both species. A comparison could then be made both within and between plant species and

a syntheis of glycophyte and halophyte differential alternative RNA splicing of the P5CS1 gene in A.

thaliana and T. salsuginea under abiotic stress could be constructed.

Future work could also include extending the analysis by using other methods of PCR technology.

Real-time PCR could be used to measure mRNA transcript levels. This would give quantitative

measurements of gene transcription in both plants under control and salt-stressed conditions. It

would provide information on how the expression of the P5CS1 gene changes overtime in response

to salt-stress (Holst-Jensen et al. 2003). Combining this with the data showing intron splicing in both

plants, this would provide quantitative and qualitative data on the abundance of P5CS1 transcripts in

both A. thaliana and T. salsuginea under control and salt-stressed conditions. The results obtained

would help to confirm and validate T. salsuginea as the plant that prepares its mature P5CS1

transcripts faster.

Other genes, such as the salt overly sensitive 1 (SOS1) have been shown to be strongly induced by

salt-stress in T. salsuginea. It functions by maintaining cellular homeostasis and osmotic balance as it

encodes a plasma membrane Na+/H+ antiporter (Kant et al. 2006). This highlights another gene that

can be targeted to ultimately enhance the salt-tolerance of crops and signifies the need to

understand the genome response to salt-stress in halophytes in order to understand their

mechanisms of resistance. Therefore, future work should focus genome wide screening to identify

and ultimately optimise the regulation of additional genes the function in responses to salt-stress.

Additionally, 60% of regulated genes have been shown to be unique to T. salsuginea in comparison

to A. thaliana (Gong et al. 2005). This suggests that both plants respond extremely differently to salt-

stress. A. thaliana was shown to employ a universal defence pathway whereas, T. salsuginea was

shown to upregulate genes functioning in post-translational modification and protein relocation

22


(Gong et al. 2005). This further highlights the need to understand whole genome responses and not

just the response of one gene to salt-stress. Future work should focus on bringing together genome

responses to salt-stress in both glycophytes and halophytes.

The promotors of the P5CS1 gene in A. thaliana and T. salsuginea are slightly different. Further

studies focusing on the promoter between both plants would help to determine if the evolution of

salt-resistance is at the promoter level. Transgenic experiments inserting the promoter of the P5CS1

gene from T. salsuginea into the P5CS1 gene of A. thaliana and comparing its growth and proline

accumulation to WT A. thaliana in saline soils would help to determine this.

The enzyme synthesised by the P5CS1 gene catalyses the rate-limiting steps of proline biosynthesis

(Mattioli et al. 2009). It is extremely important in proline accumulation as studies knocking out the

P5CS1 gene in A. thaliana have shown those plants to accumulate significantly less proline when

salt-stressed (Yu et al. 2012). However, the gene is limiting the production of the enzymes and

subsequently the biosynthesis of proline. Future work should focus on optimising the regulation and

expression of both the P5CS1 and P5CS2 (duplicated gene in A. thaliana) genes in order to maximise

the production of delta1-pyrroline-5-carboxylate synthase 1 and subsequently proline biosynthesis.

More P5CS1 transcripts would result in more proline synthesising enzymes. This would enhance the

response glycophytes show to salt-stress, optimistically enabling in the near future the growing and

cultivation of crops in saline soils.

Kesari et al. (2012) showed proline accumulation to vary among A. thaliana strains which presents

the possibility of breeding resistant strains of crop plants to produce more proline. This could be

carried out using the same methods as Borlaug used in the 20th century and highlights the

importance of retaining and utilising all possible methods to increase crop yields. This would involve

screening for varieties of crops that have higher levels of proline accumulation or enhanced

efficiency in leaf/root splicing of their P5CS1 and other stress-response genes. Crossing these plants

would result in progeny that increased the speed at which they prepare their mRNA and therefore

respond more rapidly to salt-stress. This could help in selection for A. thaliana and crop ecotypes

that do not contain the harmful exon 3 skip mutation which reduces the level of proline

accumulation and limits the ability of glycophytes to respond to salt-stress (Kesari et al. 2012). This

would enhance the response crop plants show to salt-stress if they contain this harmful mutation.

Finally, targeted screening of transcription factors, coactivators, histone acetylases and other

potential key signalling elements, such as the protein kinases ORG1 may also aid in the elucidation of

the mechanisms involved in regulating the P5CS1 gene and the general stress-responses in both

plants (Nishimura et al. 2005). Understanding the relationship the P5CS1 gene has with its

23


transcription factors and other signalling elements may reveal differences between the P5CS1 gene

in A. thaliana and T. salsuginea. This could in turn reveal different modes of regulating the gene

between both plant species and may expose the causes of the differences in the abilities of both

plants to withstand salt-stress.

5.4. Conclusion

This project presents a new field of molecular botany that can be developed in order to ultimately

enhance C3 and C4 crop regulation of stress-response genes. The P5CS1 gene remains an important

part of an interconnected and highly regulated response to salt-stress in plants. If the regulation of

P5CS1 can be optimised in theory, crops that can better regulate their stress-responses could be

produced. This would result in higher yields with no genetic modification of the coding sequence.

This would avoid the overall European stigma of GM crops while maximising crop yields and feeding

the world’s ever growing population.

The results obtained confirm proline accumulation to be more efficient in T. salsuginea and to be

characteristic of halophytic plants. Clear and distinguishable qualitative data has confirmed intron-

mediated splicing of the P5CS1 gene to be precisely regulated, controlled and different both

between and within plant species. The results provide preliminary evidence of salt-resistance being

partially due to differential intron-mediated alternative RNA splicing in the leaves and roots between

glycophytes and halophytes.

There are three main conclusions to be taken from the results. Firstly, salt-stress induces the P5CS1

gene in both A. thaliana and T. salsuginea. Secondly, Salt-stress enhances intron splicing in the

leaves and roots of A. thaliana and enhances splicing in the roots only of T. salsuginea. Thirdly, T.

salsuginea has optimal splicing in the leaves even under unstressed conditions. This advantageously

gives T. salsuginea the capacity to accumulate proline faster than A. thaliana. This protects T.

salsuginea from the harmful effects of salt-stress and enables it to grow in saline soils. All three

conclusions have aided in the molecular and physiological understanding of why T. salsuginea is a

halophyte and why it mounts and immediate response to salt-stress.

However, many questions still remain in regards to the regulation and adaptive value of the P5CS1

gene. To answer these questions extensive investment in both capital and time is required in order

to come to a more conclusive culmination of the impact of salt-stress on whole genome regulation in

plants. Further research is still needed to be undertaken before the regulation and importance of the

P5CS1 and other stress-response genes are fully understood. Answering these questions will open

vast opportunities for agriculture and biotechnology when aiming at alleviating the growing world

biofuel, feed and most prominently food insecurity.

24


6. Acknowledgements

I would firstly like to extend my thanks and gratitude to my supervisor, Dr Tahar Taybi for the

continual guidance, support and encouragement he has given me throughout my research. His

expertise and support have proven to be vital to my research.

I would like to give thanks to the laboratory technicians in the School of Biology, Mrs Roselyn Brown

and Mrs Miriam Earnshaw. Their support was essential to my overall understanding and successful

completion of laboratory techniques.

Finally, I wish to thank Newcastle University and in particular the School of Biology for giving me the

opportunity to carry out this research project.

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Cabot C, Sibole JV, Barceló J, Poschenrieder C (2014) Lessons from crop plants struggling with

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Chang L, Guo A, Jin X, Yang Q, Wang D, Sun Y, Huang Q, Wang L, Peng C, Wang X (2015) The beta

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integrative analysis of the structural, physiological and proteomic changes in chloroplasts in

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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


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.

CTTCCACGGCGTTTCCTCAGCCGCCGATTTTATTTATTTCCCAAAATACCCATCACCTATAGCGCCACAATCCTCTACATCACACCCTAATCTCATTACCATACACCACCCAACGAACACGCGCCACTTCATTTGTTAGTATCTAAAATACCAAACCTACCCTTAGTTCCACACGTGGCGTTTCCTGGTTTGATAACAGAGCCTGAGTCTCTGGTGTCGCTGGTGTTTATAAACCCCTTCATATCTTCCTTGGTGATCTCCACCTTTCCCTCACCTGATATTTATTTTCTTACCTTAAATACGACGGTGCTTCACTGAGTCCGACTCAGTTAACTCGTTCCTCTCTCTGTGTGTGGTTTTGGTAGACGACGACGACGATAATGGAGGAGCTAGATCGTTCACGTGCTTTTGCCAGAGACGTCAAACGTATCGTCGTTAAGGTTCGTTGAGATACGTTCGCATTTTCAGATTTTGTTGTTGATGATTAGATTCTTAATTTGTGATAATGTGGAAATGAATATTATGTAATTTAAGTGCATCTAAACTCTTTGTTTATTGAATTCGTGAATCTGAATATATTTTCTAATCCCAGAAACTAA

28

Table 4. RNA concentrations (ng µl-1) of extracts from 4-week-old A. thaliana and 6-week-old T. salsuginea

used in the RT-PCR. RNA extracted from the leaves and roots from water controls and plants subjected to 100

mM of NaCl for 3 days. RNA samples were read spectrophotometrically at 260/280 nm on the NanoDrop Lite

(Thermo Scientific, UK).

Table 5. RNA A260/A280 values of extracts from 4-week-old A. thaliana and 6-week-old T. salsuginea used in

the RT-PCR. RNA 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 conditions

Leaf RNA Concentration

(ng µl-1)

Root RNA Concentration

(ng µl-1)

A. thaliana T. salsuginea A. thaliana T. salsuginea

Water control100 mM Nacl for 3 days

334.9427.9

433.7534.4

602.0813.0

963.91152.1


AACTTCTCGTATGAATCTTAATTTGCATGTCATTAGAGACGAATGAATAATCAGAATATTCGAGGGATTTTTTTTCTGTTTGGTGATTAAAATTTTGGATTTTTGTTTATATTATGTAAAAAAAAAAAGGTTGGGACAGCAGTTGTTACTGGAAAAGGTGGAAGATTGGCTCTTGGTCGTTTAGGAGCACTGTGTGAACAGGTAATTGTCAAATTTTAATAATCTCCTTTTTGTATTGTGTTTATAAAAAAGTGTAAAGGTTTCATTTTTTTTCACGAAAGACATGTGAAATTATTCATGCGTAGTGGCAACTTTAATTTGTAAAAAAATATATATATATAATGTCAGCTTGCGGAATTAAACTCGGATGGATTTGAGGTGATATTGGTGTCATCTGGTGCGGTTGGTCTTGGCAGGCAAAGGCTTCGTTATCGACAATTAGTCAATAGCAGGTTAAAGCTTAATGGCTACACTTCATTATTAATCCCTTTCCCTTATAACAACATTTGGAAACAAAAAAAAAAGGGTGATGATGGATGGACCATTTTGGCTTATGTTTTTATTGCTCAATAACAGTGACATGTGTTTATGTGTGTTATGATTTAAAAGTTTTGTTTTTTTTTGCTGATGGATTTGTTTTTTTTCTTTTTTTTTGTTAATGGCTTTTGCAGCTTTGCGGATCTTCAGAAGCCTCAGACTGAACTTGATGGGAAGGCTTGTGCTGGTGTTGGACAAAGCAGTCTTATGGCTTACTATGAGACTATGTTTGACCAGGTGATTTTTCCTTTGTTATCGAATTCTAGATTATTGTGTAAGACATCCAAATATTGATGCTGTTGTTTTTCTTTGGTTAGCTTGATGTGACGGCAGCTCAACTTCTGGTGAATGACAGTAGTTTTAGAGACAAGGATTTCAGGAAGCAACTTAATGAAACTGTCAAGTCTATGCTTGATTTGAGGGTTATTCCAATTTTCAATGAGAATGATGCTATTAGCACCCGAAGAGCCCCATATCAGGTTTGTCCCTTTTGACATGAACTTTTCTACACACTCTGAGATGTGAGGGATTCTTTGAATCTCGTAGTCTAATGTTCAGCTTCACTGGATCTTGATATATGCAGGATTCTTCTGGTATTTTCTGGGATAACGATAGCTTAGCTGCTCTACTGGCGTTGGAACTGAAAGCTGATCTTCTGATTCTTCTGAGCGATGTTGAAGGTCTTTACACAGGCCCTCCAAGTGATCCTAACTCAAAGTTGATCCACACTTTTGTTAAAGAAAAACATCAAGATGAGATTACATTCGGCGACAAATCAAGATTAGGGAGAGGGGGTATGACTGCAAAAGTCAAAGCTGCAGTCAATGCAGCTTATGCTGGGATTCCTGTCATCATAACCAGGTGAGGAACCTTCTAAGCTCACCATGCATAATGATAGGGTGATATGCTTGTTCAAATTTGGTTAGATGGTATATTGATATCTTTCTTGCTTCTGAAGTGGGTATTCAGCTGAGAACATAGATAAAGTCCTCAGAGGACTACGTGTTGGAACCTTGTTTCATCAAGATGCTCGTTTATGGGCTCCGATCACAGATTCTAATGCTCGTGACATGGCAGTTGCTGCGAGGGAAAGTTCCAGAAAGCTTCAGGTAATTGTGACTTATGCATGGCTTTCTTTCATGTTCGTAACGTCAAAAACCATTCTTGCTCGGCATAGAGTTACTTAACTTTTTTTTACATTTTGCTATAGGCCTTATCTTCGGAAGACAGGAAAAAAATTCTGCTTGATATTGCCGATGCCCTTGAAGCAAATGTTACTACAATCAAAGCTGAGAATGAGTTAGATGTAGCTTCTGCACAAGAGGCTGGGTTGGAAGAGTCAATGGTGGCTCGCTTAGTTATGACACCTGGAAAGGTAAGAAAGTATTCATGGCCATAGATAGTTGCTTTTTGTTGCTATGGCTTGGGCAAACATATTGTGCCAATGTAACCTCTCCTTATTATGTTTCTTATTTTGTGCTTGATAGATCTCGAGCCTTGCAGCTTCAGTTCGTAAGCTAGCTGATATGGAAGATCCAATCGGCCGTGTTTTAAAGAAAACAGAGGTGATCAGAGGACAATTGTTACCATATAGTTAATTTACATACTCTTGAGTTAAATAAGGGATATGACTATCCTCCTAGTTGACATACAATAGTTGTTTATGCTATTTGTTCTTTGTGGCAATTCCTTTTACAGGTGGCAGATGGTCTTGTCTTAGAGAAGACCTCATCACCATTAGGCGTACTTCTGATTGTTTTTGAATCCCGACCTGATGCACTTGTACAGGTATGTTAATAGTCAAAATTCATTTCCCTTCTTAATATGTGAATTTCCTAAAGCTGTGCTTTATCCACAAACCAAACAGATAGCTTCACTTGCCATCCGTAGTGGAAATGGTCTTCTGCTGAAGGGTGGAAAGGAGGCCCGGCGATCAAATGCTATCTTACACAAGGTACCATTGCCTCAGATTTCATATCATTATTTGCCTCAAAATTTATCACTACAGCTCTTTTAAGTTCATGGTAAATTTCTAGGTGATCACTGATGCAATTCCAGAGACTGTTGGGGGTAAACTCATTGGACTTGTGACTTCAAGAGAAGAGATTCCTGATTTGCTTAAGGTAAGAACAGATTTACAAGCTAGGAGCTGCAACAGTTCTTTTGTATCTTTTGTTAAACTGGAACCCACCATTTGCATTTGTGTTACAGCTTGATGACGTTATCGATCTTGTGATCCCAAGAGGAAGCAACAAGCTTGTTACTCAGATAAAAAATACTACAAAAATCCCTGTGCTAGGTCATGCTGGTATGGTTGCAAGTTTGTTTTTTCCAGAAGATTCTTTACTTGGATTGTGCTAGAGTGTGACGATGGCTTAATTGTGTACTTGCAGATGGAATCTGTCATGTATATGTCGACAAGGCTTGTGATACGGATATGGCAAAGCGCATAGTTTCTGATGCAAAGTTGGACTATCCAGCAGCCTGTAATGCGATGGTAAGAGAACTTTTTACCTTCCATCGAGATTTAATTAATACAGTGGGAGATTCTAAAGTTCAACTGACTCATTTCATCTTCTCTCGTCTCTTTCAGGAAACCCTTCTTGTGCATAAGGATCTAGAGCAGAATGCTGTGCTTAATGAGCTTATTTTTGCTCTGCAGAGCAATGGTACGTCATAAATGGCCCAATCATTTGTTGGTCTATCTTAACCATTTATTTGACCTCTTGTTACCTTCCATCTGGATGTCTCATAGATATACATGTAGCCTGTTTGATTATAAATATTGAATGGTCATCTCATGAAAACATTTCTAGAGTGGCATAACTCATGAGATATATTAAACTACAGGAGTCACTTTGTATGGTGGACCAAGGGCAAGTAAGATACTGAACATACCAGAAGCACGGTCATTCAACCATGAGTACTGTGCCAAGGCTTGCACTGTTGAAGTTGTAGAAGACGTTTATGGTGCTATAGATCACATTCACCGACATGGGAGGTAGAAAC

29


TCGACATAACAGGCATTGACTTTAGAAATTCTTTGCATATGTAGTGGAAATGTTCACTCGTTATCTTGTCTTGTATGTTGTTACGAGCAGTGCACACACAGACTGCATTGTGACAGAGGATCACGAAGTTGCAGAGCTATTCCTTCGCCAAGTGGATAGGTAAAGTACTGAATCTTTAACTTGCTTATTATCTGTCTTTGATTTTTCTTGGAAACTGACTGTAAGATGTTGCGACCTTGAACAGCGCTGCTGTGTTCCACAACGCCAGCACAAGATTCTCAGATGGTTTCCGATTTGGACTTGGTGCAGAGGTAAGTCAGAGACATACACATAAGTCTATAGATTAAAAACAAATAAAAAGAGGAAGAGTGAGTGATAAAAAAGTATTGGTTGTGGTATATAGGTGGGGGTAAGCACGGGCAGGATCCATGCTCGTGGTCCAGTCGGGGTCGAAGGATTACTTACAACGAGATGGTACAATTTTAGTTACTCAAAGCACCATTGTTATGTCAATAAAGACCCACAATAAGCCTTTTTTCCTATGCTTCTTTTAATTTTCATGGTGAAATGGTTGCAGGATAATGAGAGGAAAAGGACAAGTTGTCGACGGAGACAATGGAATTGTTTACACCCATCAGGACATTCCCATCCAAGCTTAAACAAGACTTCCGAGTGTGTGTTTGTGTATTTGGTTGAGACTTGAGGAGAGACACAGAGGAGGATGGGCTTTTTTGTTTCCTCTCTGCTTAGTACTCATATCCTATCATTATTATTATTACTACTACTTATTATTGAAACCCTCGCTTATGTAGTGGTTTTGATTTAGGGTTAGGATTGCACCAAAAATAAGATCCACTTTACCACTTAGTCTTGCTCATAAGTACGATGAAGAACATTTAATTAGCTTCTCTTCTTGTCATTGTAAGCTACCTACACATTTCTGATCTTTATCAAGATACTACTACTTTTCATTTCGCTTATCTATAAATATATTTCGATTTGCATTGGAAATCACAAGTTGAATCAGAACTGGAAACTCTTAACCATAAATTCTCAAAGATTGTGCTACATTTGAAAGCTAACAATGAACACAAGAAAAGAAC

(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.

GACACTTCCCTCACCAGATATTTCCCTAAACGCGCTCACTGACGAAATCCACCACTGAGTTAACTCGTTCCTTCTCTGGGTTTTGGTAGGCGGCGACAATGGAGGAGCTAGATCGTTCACGCGCTTTTGCCAAAGACGTCAAGCGTATCGTCGTTAAGGTCTCGTCTCTCTCTCTCTCTCTCTTTGTATCTGTTTGTTTATCTCCTTATCCGTGTTTCGTTGAGAAACGTCCGCATTCTCAGATTTTGATTTGATTATCGACTGTTTTTGGCTTAATTGCTGATTTCGATTTTTTTTTGTTTTTTTTTCTCTGCGTTCGTCTGAATCTGTGAAGTGTTCGTCGTCGTTGGTTGTCGATGTGGATTGGGTTTAGTGTGTTTTTTAATTTCATTTTAAGCTGTTTTTTGCGGCTGAGTGAAATCTGCGGTAATGTGAAAAATCGAATATTATATGATTTAACGTGCATCTGAATATTTTTTGTTTGTCTCTGTTATTGAAAAGCTCTCAACGGAAAAGTTTCTCGAATCTGAATACCATTTGTCTCGGAAAAATTAAACCTCTCGTAATCACGCTTATGAATCTTAATCTGCATGTCATCAG

30


AGAGTGATGAAGAATCAGAATATTCGGATAATTAATATTCTGTTTTTTTTTTTTTGTAAATATAGGTTGGGACCGCTGTTGTTACTGGGAAAGGTGGAAGATTGGCTCTTGGTCGCTTAGGAGCACTGTGTGAACAGGTATTTTGATTTTTATTATTTACCTTAATTATCATTAACCTATGTTAATTAATCAGCTTTTTGCTTTATTCCTAAATTGTGTAAAAAGGTTTCACGAAATACATGTGATGCAATTTTGCACCTTTAATTCGTAAAATATATATTATAATGTCAGCTTGCGGAATTAAACTCGGATGGATTTGAGGTGATTTTGGTGTCATCTGGTGCGGTTGGCCTTGGCAGGCAAAGGCTTCGATACAGACAATTAGTCAATAGCAGGTTAAGCAAAATGGCAACTTTTAAACCAATCATTTCACTTTAATCTTATTGGAATCAAAAAGGGTGATGGACCATTGACTTATGTTTGCTTTCTGATGGGAATAACAGTGAGATGTGTTTATGATTTTAAAGTTTTTGTTTTGTGCTGAGTTTATTTCTTAATGGATTGCAGCTTTGCGGATCTTCAGAAGCCTCAGAGTGAACTTGATGGGAAGGCTTGCGCTGGTGTTGGACAAAGCAGTCTTATGGCTTATTACGAGACTATGTTCGACCAGGTGATTTTTCTCTTCTTTTTTTAAGGAAGAAGACTATATATGGTCTCGTTTTCTTAATTGCTGTGTAAAATTCCAAATATTGATGCTTTGTTTCCTGTTGTTTTCTTTGGTCAGCTGGATGTGACGGCGGCTCAACTTCTGGTGAATGACAGTAGTTTTAGAGACAAGGATTTCAGAAAGCAACTTAATGAAACTGTCAAGTCGATGCTTGATTTGAGGGTTATTCCGATTTTCAATGAGAATGATGCTATAAGCACCAGAAGAGCCCCATATCAGGTTTGTTGACTATCTTTGGTCCCTTTGAAATGAGTACTCCTTTGAATTTAGCTGCTTCCTATGAATCTCGTAGTCTTATATGTTCAACTTCATTGCATTTCAATATACGCAGGATTCCTCTGGCATCTTTTGGGATAACGACAGCTTAGCTGCTCTACTGGCGCTGGAACTGAAAGCTGACCTTCTGATTCTTCTGAGTGATGTCGAAGGTCTTTACACAGGCCCTCCCAGTGATCCTAACTCAAAGTTGATCCACACATTTATTAAGGAAAAACATCAAGATGAGATTACATTTGGCGACAAGTCAAGATTAGGAAGAGGTGGCATGACTGCAAAAGTCAAAGCTGCAGTGAATGCAGCATATGCTGGGATTCCTGTCATCATAACCAGGTGTGGGCCCTTTTACATTCATTGTGCATAATTAATACGCTTTCCAAATTTGTCAAGTGTTTTTGATCTCGCTTTCAGTTCTGACCCTGAATATCATCATCTTAATTCCTCCGAAATACCACAATTTACGTTTGATTGAGAAATATTCGAAAGATATTTTGTTGGATAGAAAGCTGATACTTTTCTTGCTTTTGAAGTGGGTATTCAGCTGAAAACATAGATAAAGTCCTCCGAGGACTGCGTGTTGGAACCTTGTTCCATCAAGATGCTCGTCAATGGGCTCCGATCACAGATTCTACTGCTCGTGACATGGCAGTTGCTGCAAGAGAAAGTTCCAGAAAGCTTCAGGTACTGCTAGTTGCTGCATGCGTATCTTTTTTCCACAATTATGATGTGAGAAATCTTTTCTTTCTCGGTAGAGATGTATTTAAACTGCTTGTAATTTGCTACAGGCCTTATCTTCAGAAGATAGGAAACAAATTCTGTATAATATCGCCGACGCTCTTGAAGCAAATGAAAAAACAATCAGAGATGAGAATGAATTAGATGTATCTGCAGCACAAGAAGCTGGATTTGAAGAGTCATTGGTGGCTCGCTTAGTTATGACACCTGCAAAGGTAAGACAGTATTCGTGTTGTGTGGTATTGTGCCAATTTCACCTCTCCTGATGATCTATATATCTTGTTTTTATTTCTAATGTTTTCTTGTTTTGCTTGATAGATCTCAAGCCTTGCAGCTTCAGTTCGTAAGCTAGCCGATATGGAAGATCCAATTGGCCGTGTTTTAAAGAAAACTGAGGTGATCAGAGGACAGTTGTTATTATATAAAGTTTTACAGTCTAGGAGTATCCTCGTAGTTGACATATAATAGCTGTTTATCCTATTCGTTCATCGTGACAATTGCTTTTACAGGTGGCAGATGGTCTTGTCTTAGAGAAGACCTCATCCCCATTAGGCGTACTCCTGATTGTTTTTGAATCCCGACCTGATGCACTTGTACAGGTATGTTAAGAGTCAATGTCCTTTTATCTTCTTAGAATGTGAATTTGCTGAAACCTGTGTTTTATCCACAAACCAAACAGATAGCTTCACTTGCCATCCGGAGTGGAAATGGTCTTCTATTGAAGGGTGGAAAGGAGGCCCGGCGATCAAATGCTATCTTACATAAGGTACAGTGCCTCAGATTTCAGACTCGGATGTTATCATATATGGCTTCCTCAAAATATGCTGGTTATAATTGATCCATTTAATTTCATTTTAAATTTCTAGGTGATCACTGATGCAATTCCAGAGACTGTCGGGGGTAAACTCATTGGACTTGTGACTTCAAGAGAAGAGATTCCTGATTTGCTCAAGGTAAACGGATTTACAAACTTGGAGCTGCAACAAATCTTTATATCTTGTGTTTAAATGGAAACCACCATTTGCATTTGCGCTACAGCTTGATGACGTTATAGATCTTGTGATCCCAAGAGGCAGCAACAAGCTTGTTTCCCAGATAAAAAATACTACAAAAATCCCTGTGCTAGGCCATGCTGGTACGGTTTCAAGTTTGTTTTTCCATAAAATTCTTTAGTTGGATTGTGTTAGAGAGTGACTGTCTTAATTTTGTACTTCCAGATGGAATCTGTCATGTATATGTCGACAAGTCATGTAATCTGGATATGGCAAAGCGCATAATTTCCGATGCAAAGTTGGATTATCCAGCAGCCTGTAATGCGATGGTAAGAGAACTTGTACCAGCCTCTTGAGATTGGAGTATGCAATGGGCGTATTAATTTCATCCGACTCATTTCACCTTCTCTTTCCTTTGTATTTTCAGGAAACTCTTCTTGTGCATAAGGATCTAGAGCAGAACGGGCTCAATGAGCTTATTTTTGTGCTGCAGAGCAATGGTATGTCATAAATGCCGTGTTTGTTGGTCTCTCGTAATCCTGAAGACTTTTTTTTTGGTTGGTAAAATTAATTCTGAAGACTTGTTTGGAGTAATTTAACTCATGAAGTATTTTTAACTGCAGGAGTCACTGTATATGGTGGACCAAGAGCAAGTGCAATACTGAACATACCAGAAGCACGGTCGTTCAACTATGAGTACTGTTCCAAGGCTTGCACCGTTGAAGTTGTAGAAGACGTTTACGGTGCTATAGATCACATTCACCGACATGGGAGGTAAAAACTCGATATAACAGACATTGAGTTTTGTAATCTTTTTGCCT

31


ATGTACTGGAAATGTTCACTCTTTATCTTGTCTTATATTTTGTTACGAGCAGTGCGCACACAGATTGCATTGTGACAGAGGATACCGAAGTTGCAGAGCTATTCCTTCGCCAAGTGGACAGGTAAAATACCGGATCATGAACTTGTTTAGTGGCTGTCTTTGATTATGTTGGTAACTGACTGTAAGATGTACGTCCTTGAACAGCGCTGCTGTTTTCCACAACGCAAGCACAAGATTCTCAGATGGGGCTCGATTTGGACTTGGTGCCGAGGTAAGTGAGAGACATACAAATAATCCTATTTATCAAACAGGGAAAAGAGGGAAGAGTGAGTGATGAAGTAAGTTTTGGTTGGTTATACATAGGTGGGAATAAGCACAGGTAGGATTCATGCTCGTGGCCCAGTCGGAGTTGAAGGATTACTTACAACAAGATGGTACCATTTTACTTACTTGAAACACCATTGTTGTTATGTCGATATATCCTCGCAATAAGCTTTTTCTTCTTAGCTTTATTTGTAAATTTTCATGGTGAAATGGTTTGAAGTATGAGTGATGGTGGTTGCAGGTTAATGAGAGGAAAAGGACAAGTTGTTGATGGAGACAATGGGATTGCTTACACCCATCAAGACATTCCCATCCAATCTTAGAAGACTGTTGTGTGTTGAGACTTGAGGAGAGGATGGGCTTTTTGTTTCCTCTCTGCTAATATCATATCCTATTATTATTGTTATTGAAACCCTCTCTTATGTAGTGGTTTTGATTTAGGAATTAGGGATTGCACCAAGAATAAGTTACCACTTGGTCTTGCTCATAAGTAAGATGAAGAACATTTTCTTAGCTTCTCTTCTTGTTTAAAAAAAACACGTTGTAAGGCTACCTACACCTTTCTGATTTATCATTTATCTATATCTTTGGATTTGAGTTTGGACTTCCACTGGGAGTTATACCTTTAATACAAAGTTGCATATATGAACTTAAAAAGTCATTACTATTAATTCCCAAGGATCAGCGCAAAATGGTAAACACGTTTGAGTACGTTTGAGTGCTAAGAATGAACACAAGAGTTCTCATTCTTACATATCTAATTTTTTTCAGGTACTTGAGGAATCGATCTTAACTTTCATTCTTCCATAAACTTCTTAACATTCTTCAACCACAGCAAGTACTCTCGCTTCACTTTTCTCATCATGTACTZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZ

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