Dissertation thesis

Newcastle University May 5, 2016
1
School of Biology
Gene 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: BiologicalResearch Project
Supervisor: Dr TaharTaybi
2015/2016
Word Count: 8000

2
1. Abstract
Crop productivity is limitedby environmental stresses including salt-stress. Proline accumulates in
leavesunderstressconditionsasanimportantosmoprotectantandanti-oxidant.The synthesisof this
important amino acid is controlled by the enzyme Delta1-pyrroline-5-carboxylate synthase which is
up regulatedatthe gene level byavariety of stresses.Inthisprojectintron-mediatedalternativeRNA
splicing as a means of regulating the P5CS1 gene was analysed under salt-stress using RT-PCR
technologyinboththe glycophyte, Arabidopsisthaliana andthe halophyte, Thellungiella salsuginea.
Results confirmed P5CS1 to be induced by NaCl and showed a significant difference in proline
accumulationbetweenthe twoplantspeciesaswell asbetweencontrol unstressed plantsandplants
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 howeversplicingof these intronswasenhancedby salt-stressinbothspecies. Spatiotemporal
regulation of the P5CS1 gene between plant organs is a likely explanation of its control due to
differentialsplicinginboththe leavesandrootsof plantswhenunstressedandsalt-stressed.The data
showsdifferentialregulationof the P5CS1gene inglycophytesandhalophyteswhensubjectedtosalt-
stress and highlights tissue specific regulation of the gene as a possible factor contributing to salt-
tolerance inhalophytes.Thisprovidespromisingapplicationsinbiotechnologyandagriculture 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.

3
Contents
Abstract 2
Introduction 4
Historyand currentdevelopmentsinagricultural botany 4
Salt-stressasa significantabioticstressor 4
Plantresponsestosalt-stress 5
Δ1-Pyrroline-5-carboxylateSynthetase 1(P5CS1) gene andproline accumulation 5
Genome regulationasafactor conferringsalt-tolerance 6
Aimsandhypothesises 7
Methods 7
Materialsandmethods 8
Plantmaterial andgrowthconditions 8
Proline determination anddataanalysis 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 andcDNA intronsplicing 14
Root cDNA intronsplicing 16
Discussion 18
Discussionof results 18
Limitations,critical appraisaland improvementstothe studymethods 20
Future work 22
Conclusion 24
Acknowledgements 25
References 25
Appendices 28

4
2. Introduction
2.1. History and current developmentsinagricultural 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 20th
century and significantly increased food production. Norman Borlaug, the father of the green
revolution,focusedon breeding crop plants that increased the biomass they portioned to the grain
(Borlaug2000). Hiswork ledto the development of lodging-resistant,highyielding,disease resistant
semi-dwarfgrainvarieties(Borlaug2000).These varietiesdoubled cropyields inlinewithanincreasing
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
cruciallylimitingresource acrossthe word,yetdemandforitcontinuestosoar.Additionally, the most
successful wheatplantsinvestapproximately60% of its resources intothe grain (Borlaug 2000). It is
unlikely thatscientistscanincrease thisanyfurther.Thishighlightsthe importance of identifyingand
developingnovel methodstoincrease cropyields. Ourplanetisfacingmore evident andpronounced
challenges thatwere notassevereduringthe lastgreenrevolutionandtogetherthesefactorsfurther
widenthe gapbetweenbotanicalsciencesand the globalfoodinsecurity phenomenon. Tomeetthese
demandsandfeedtheincreasingworldpopulationa70% increase inglobalfoodproductionisneeded
by 2050, whichincludesanadditional 1billiontonnesof cereal crops (FAO2009).
2.2. Salt-stressas a significantabiotic stressor
Sodiumsaltsdirectlyimpactthe survival of landplants.Ourmostvaluedterrestrial plants,the cereals
are classified as glycophytes and are particularly vulnerable to salt-stress as they die at salt
concentrationsof approximately100mM NaCl (Munnsand Tester2008). Whereas,halophyticplants
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
supplyisproducedandsoilsalinityisexpectedtoresultin50% of arable land tobe lostby2050 (Wang
et al.2003). Due to this,extensive research hasbeencarriedoutoverthe last20 yearsto understand
mechanisms of stress-tolerance in order to develop crop plants that can survive in extreme salt
concentrations. Thispresentsapossible fieldof scientificmanipulationthatcan aid in the alleviation
of the global foodinsecurity challenge withoutcroplandexpansion.

5
2.3. Plant responsesto 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,transductionandtranscriptional up- ordown-regulation
is the production of proteins and molecules with various plant protection, repair and stabilisation
functions,suchasthe osmoprotectantaminoacidproline(Gongetal.2005).These mechanismsadjust
the osmoticpressure backtooptimal levelsinordertomaintainwater uptake,cell turgorandgrowth
(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
genesthatsynthesisekeyproteinsinvolvedinstress-responsesandoptimisingtheirregulationincrop
species. This will help science to produce crops that can survive and grow in saline environments,
helpingtooffsetfoodinsecurity.
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 RNAsplicingof the intronsintheA.thalianaandT.salsuginea P5CS1
gene are analysed inthisreport.P5CS1 encodesthe 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 andsubsequentlyinduces expressionof the genethroughoutthe wholeplant (Yoshibaetal.
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 asacompatible osmolyteandacryoprotectant butitsregulationandadaptiveimportance are
yetto be fullyconcluded (VerbruggenandHermans2008). Differential expressionundersalt-stressin
A.thaliana andT. salsuginea have beenshowntocorrelatewithhigher P5CS1transcriptlevels,higher
levelsof prolineinthe leaves andenhancedcontrol overNa+
uptake in T.salsuginea(Kantetal.2006).
This was furtherexploredinthe project.However, furtherresearchis neededtoconfirmthe factors
regulatingthese responses.

6
2.5. Genome regulationas a factor conferringsalt-tolerance
The ability of plants to respond optimally to salt-stressis 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
widelyrecognisedthroughextensive researchintothe mechanismsof salt-tolerance thatdifferential
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 etal. 2012). The halophyticand glycophyticregulationof the P5CS1 gene will be
considered throughout this report with a consideration of the possible practicalities of applying the
resultsobtainedtoC3 andC4 crops.
T. salsuginea hasbeenshowedtocontainhigherlevelsof proline whenunstressed,andwhenstressed
itsynthesisesmore proline than A.thaliana (Kantetal.2006). Manyhypothesisesof the salt-tolerance
in T. salsuginea have been described. Firstly, the ortholog of the proline degradation enzyme in A.
thaliana (PDH) hasbeenshownnottobe expressedand isundetectable intheshootsof T.salsuginea,
indicatingprolinecatabolism isstronglysupressed (Kantetal.2006). A higherbasal level of proline is
thoughttoaidinthe responseT.salsugineashowswhenexposedtosalt-stress.This isbecause ithelps
T. salsuginea mountan immediate and efficientresponse tothe stressor.Sequencingthe genomeof
T. salsuginea alsoshoweditto have a similarexonlengthto A.thaliana but a far largerintronlength
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 overitsstress-responsegenes. The resultsobtainedbyWuetal.(2012) were furtherexplored
and builton in this project.These factors highlightthe importance of understandingthe modulation
of the transcriptome and proteome at the transcriptional and post-transcriptional level under salt-
stressconditionsbetween A.thaliana andT.salsuginea.Thisisbecause understandingthe regulation
of P5CS1 may aidin the elucidationof the mechanismsandkeyregulatorsinvolvedinthe production
of adequate physiological responses and their evolution in different plant systems. The knowledge
gainedfromthis may be used in the productionof crop varietieswithanenhancedtolerance tosalt-
stressthat can be grown inpreviouslyinhabitableenvironments.

7
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
inducedbysalt-stressandif there wasadifference between A.thaliana andT.salsuginea?Focuswas
on intron-mediated alternative mRNA splicing of the P5CS1 gene as a possible contributor to the
highersalt-tolerance shownby T.salsuginea comparativelyto A.thaliana.Resultsshow the response
to salt-stressat the tissue level betweenandwithinbothspeciesandprovide some preliminarydata
that beginsto uncoverhalophyticandglycophyticregulationof the P5CS1 gene.The projectfocused
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 betweenthe splicingof the intronsundercontrol andsalt-stressedconditions
between A. thaliana and T. salsuginea in both the leaves and roots. It was hypothesised that T.
salsuginea preparesitsmature transcriptsignificantlyquickerthan A.thaliana inthe leavesandroots
and thatintron-mediatedsplicingisworkingatfull speedinbothcontrol andsalt-stressedconditions.
This would mean that unlike A. thaliana, T. salsuginea mounts an immediate response to salt-stress
whichconfersitsresistance tothe abioticstress.
2.7. Methods
Methods to obtain the results include: gDNA (leaves) and RNA (leaves and roots) extractions from
control andsalt-stressedplants.The gDNA samples wereextractedfromthe leavesof bothplants and
were used to confirm the complete set of introns were present in both plant species whenexposed
tocontrol conditions (unstressed).Qualitative RT-PCRwasperformedonthe RNA extractedfromboth
the water control plants and plants subjectedto 100 mM NaCl for 3 days. This method was used to
reconvertthe mRNA to cDNA fromthe watercontrol andsalt-stressedplantsof bothspecies.Agarose
gel-electrophoresiswasusedtorunthe samplesinordertoconfirm the presence of the intronsof the
P5CS1 gene inbothplantsinthe gDNA controls of bothspecies.Italsoenabledthe comparisonof the
splicingof intronsinthe codingregionof P5CS1inboththe watercontrol andsalt-stressedconditions
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 bothwithinandbetweenspecies.Agarose gel-electrophoresiswas the bestmethodtouse as it
allowed the experimenter to easily compare the response to salt-stress between and within plant
speciesandtissues.

8
3. Materialsand methods
3.1. Plant material and growth conditions
A. thaliana (Columbia ecotype) and T. salsuginea (Shandong ecotype) seeds were surface sterilised
using70% ethanol,washedthreetimeswithsterile waterandsownonJohnInnessoilcompostNo.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
intensityof 150μmol.m-2
.s-1
atplantheight.Seven-day-oldseedlingswerethentransferredtosmaller
pots(2.5 cmwide) containingmoistJohnInnesNo.3compostwithoneseedlingineach.Then4-week-
oldA.thaliana and 6-week-oldT.salsuginea plants,similarinsize andbeforebolting,wereseparated
intothree setsandirrigatedwiththreedifferentNaCl concentrationspreparedwithnormaltapwater.
A.thaliana was wateredwith0,and100 mM[NaCl] and T. salsuginea waswateredwith0,100 [NaCl]
(0 mM refers to tap water) at a fixed time (12:00) every day for 10 days. Shoots and roots were
harvestedatafixedtime (16:00) asthree plantspersample after3daysof the salttreatment,weighed
and frozen in liquid nitrogen. Three samples were harvested at each time point for each NaCl
concentrationforbothplantspecies.Control plantswere wateredwithtapwateronlyandharvested
inparallel tosalt-treatedplants.
3.2. Proline determination
Nine plantsintotal were grownandleaf samples (secondleaf fromthe shoottip) fromthree 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 (Batesetal.1973) but optimisedtothe specificsof thisexperiment.Volumesandmassesof
ninhydrin were based on those used by Claussen (2005): 2.5 g ninhydrin/100ml consisting of glacial
acetic acid, sterile water and 85% ortho-phosphoric acid in proportions of 6:3:1 (Claussen 2005). 10
ml of 3% (w/v) aqueoussulfosalicylicacidandquartz sandwasaddedto 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 thenusedto filterthe homogenate.The remainswere discardedandthe
clear filtrate was usedinthe proline assay. 1 ml of ninhydrinandglacial aceticwere addedto1 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

9
calculated based on the μmol of proline per g of leaf fresh weight (μmolproline (gFW)−1
) (Claussen
2005).
3.2.1. Data analysis
There wasno significantdeviationbetweenthe variancesof the residualsandnormal distributionfor
bothA.thaliana andT. salsuginea. Therefore, agenerallinearmodelwasusedtomodelthe effectsof
plantspeciesandsalt-stressonprolineaccumulation.
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
addedto a 1.5 ml tube and 100 mg of groundplant tissue wasadded tothis.5 µl of proteinase Kwas
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 spinfilter and spun in the same conditionsas before.The filtrate
wasdiscardedandplacedona spinfilter onareceivertube andaddedtoitwas550 µl of washbuffer
I before beingspunagaininthe same conditions.Thisstepwasrepeatedagainbutthistime with550
µl of wash buffer II. The filtrate was discarded and the spin filter was placed on a receiver tube and
spuninthe same conditionsagainbutthistime todryout the resininthe spinfilter.The productwas
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 lefttostandfor 2 minutesatroombefore beingspuninthe same conditionstoelute the gDNA.
3.4. Qualitative DNA PCR
The followingreagentswereaddedtoPCRtubestomakea25µl reaction:1µl gDNA (Table 3) orcDNA,
1 µl of the forwardprimer (10 µM), 1 µl of the reverse primer(10 µM), 12.5 µl x2 MyFI Mix (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 annealingtemperature wasoptimisedto58°C for30 secondsandthe extensiontemperature 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 lidtemperature wassetto105°C.Sampleswere eitherusedimmediatelyorstored
at -20°C.

10
3.5. RNA extraction
Followingthe TRI-REAGENTmethod,plantmaterialwasgroundtoafine powderusingliquidnitrogen
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
minutesandthenspunat 13000 rpmat 4°C for 10 minutes.The upperphase wasthentransferredto
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,
vortexedandthenspunat 13000 rpm at 4°C for 5 minutes.The supernatantwasremovedagainand
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 (ThermoScientific,UK) anddisplayedinTable 4and 5. RNA was extractedfrom3 differentplants
and mixedtogetherforeachcondition andDNase treatedbefore the RT-PCR.
3.6. Qualitative RT-PCR
Using the Tetro cDNA SynthesisKit(Bioline,UK) RNA was reverse transcribedtocDNA. 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
preparedinan RNase-freePCRtube asfollows:5 µl of RNA persample wasadded andthe restfrozen
at -80°C for long term storage. 1 µl of the oligo (dT)18 primer, 10 mM dNTP mix, RiboSafe RNase
inhibitorandthe TetroReverse Transcriptase (200u µl-1
) wasthenaddedtothe same tube.4µl of the
5x RT bufferwasaddedand finally7 µl of DEPC-waterwas added to bring the total volume 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 storedat -20°C for long
termstorage.
Table 1. The sequences of each primer base pair andpredictedamplicon size for both unsplicedandsplicedintrons of the
P5CS1 coding sequence in Arabidopsis thaliana. Ampliconsizes (bp)were calculated for introns 1-20. Introns 6 and7 were
amplifiedas a single amplicon. Primers from Integrated DNATechnologies, Belgium.

11
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
20
5’ – TCG TTA AGG TTC GTT GAG
– 3’
5’ – GAT TGG CTC TTG GTC GCT
TA – 3’
5’ – CTT GCG GAA TTA AACTCG
GAT G – 3’
5’ – AAG CCT CAG AGT GAA CTT
GAT G – 3’
5’ – CTC AACTTC TGG TGA ATG
ACA G – 3’
5’ – CCT AACTCA AAG TTG ATC
CACAC – 3’
5’ – ATA GAT AAA GTC CTC CGA
GGA C – 3’
5’ – TAT AAT ATCGCC GACGCT
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 ACTTGC
CAT C – 3’
5’ – TGC CATCCG 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 AACTCT TCT TGT GCA
TAA GG – 3’
5’ – TCA CTG TAT ATG GTG GAC
CAA G – 3’
5’ – CACACA GAT TGC ATT GTG
ACA G – 3’
5’ – TTT TCC ACA ACG CAA GCA
CAA G – 3’
5’ – GTC GGA GTT GAA GGA TTA
CTT AC – 3’
5’ – ACG ACCAAG AGC CAA
TCT TC – 3’
5’ – GAC TAA TTG TCT GTA
TCG AAGC – 3’
5’ – CGA ACA TAG TCT CGT
AATAAG CC – 3’
5’ – CTC TTC TGG TGC TTA
TAG CAT C – 3’
5’ – GTG TGG ATC AACTTT
GAG TTA GG – 3’
5’ – GTG AAA GTT CCT AGA
AAG CTT AG – 3’
5’ – AAG AGCGTC 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 AACAGT
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
ACT CCG AC – 3’
5’ – TCC TCA AGT CTC AAC
ACA CAA C – 3’
347
281
223
318
396
351
288
347
270
204
257
217
211
244
150
302
200
179
179
59
134
107
238
293
250
191
229
137
71
171
135
126
159
69
204
115
87
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
18
19
5’ – CTT CCC TCA CCA GAT ATT
TCC – 3’
5’ – ATT GGC TCT TGG TCG CCT
AG – 3’
5’ – CTT GCG GAA TTA AACTCG
GAT G – 3’
5’ – AAG CCT CAG AGT GAA CTT
GAT G – 3’
5’ – CTC AACTTC TGG TGA ATG
ACA G – 3’
5’ – CCT AACTCA AAG TTG ATC
CACAC – 3’
5’ – ATA GAT AAA GTC CTC CGA
GGA C – 3’
5’ – TAT AAT ATCGCC GACGCT
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 ACTTGC
CAT C – 3’
5’ – TGC CATCCG 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 AACTCT TCT TGT GCA
TAA GG – 3’
5’ – TCA CTG TAT ATG GTG GAC
CAA G – 3’
5’ – CACACA GAT TGC ATT GTG
ACA G – 3’
5’ – TTT TCC ACA ACG CAA GCA
CAA G – 3’
5’ – GTC GGA GTT GAA GGA TTA
CTT AC – 3’
5’ – AGT GCT CCT AAGCGA
CCA AG – 3’
5’ – TCT GTA TCG AAG CCT
TTG CC – 3’
5’ – CGA ACA TAG TCT CGT
AATAAG CC – 3’
5’ – CTC TTC TGG TGC TTA
TAG CAT C – 3’
5’ – GTG TGG ATC AACTTT
GAG TTA GG – 3’
5’ – GTG AAA GTT CCT AGA
AAG CTT AG – 3’
5’ – AAG AGCGTC 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 AACAGT
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
ACT CCG AC – 3’
5’ – TCC TCA AGT CTC AAC
ACA CAA C – 3’
723
278
374
352
404
503
290
300
250
184
275
235
211
155
287
303
190
213
273
216
124
202
238
293
306
187
197
137
71
171
135
126
73
162
204
107
121
142
Table 2. The sequences of each primer base pair andpredictedamplicon size for both unsplicedandsplicedintrons of the
P5CS1 coding sequence in Thellungiella salsuginea. Amplicon sizes (bp) were calculated for introns 1-19. Primers from
IntegratedDNA Technologies, Belgium.

12
3.7. Agarose gel-electrophoresis
1.5% agarose gelswere made byweighing 3g of agarose (Moleculargrade) andputin a conical flask.
200 ml of x0.5 trisboricacidEDTA (TBE) wasaddedtothisandthenthe contentsswirledtomix them.
The agarose was thenmeltedininmicro-waive andonce meltedit wasleftto cool.Once cooledand
whenwearingglovesandgoggles9µl of EthidiumBromide(stocksolution) wasaddedandthe conical
flaskswirled.The agarose solutionwasthenpouredintoapre-preparedgel trayandlefttosolidifyfor
30 minutes.The gel wasthenplacedinagel box and submergedinx0.5TBE. 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) foreachof the 19/20 intronsstudied.Sampleswere runfor1hourat 100 V andthen
gelswere visualisedunder UV lightusingagel-docsystem.
4. Results
4.1. Proline accumulation
Both plantspeciesand salt-stress(Figure 1) hada significanteffectonproline accumulation (ANOVA,
Plantspecies: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
regressionequationwas 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 (gFW)−1
in T. salsuginea when exposed to 100 mM of NaCl for 3 days. This suggests that plant species has a
slightlystrongerinfluence onproline accumulationthansalt-stress(whenmeasuredin μmol (gFW)−1
)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
A. thaliana T. salsuginea
ProlineConcentration
(μmol(gFW)−1)±s.e.
Plant Species
3 days water 3 days 100 mM NaCl
Figure 1. Proline concentration (μmol proline (g FW)−1) in Arabidopsis thaliana and Thellungiella
salsuginea subjectedto control (3 days of water) and salt-stress (3 days of 100 mMNaCl) 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.

13
althoughbothfactorshave showntoeffectproline accumulation similarly.Figure1shows thatproline
concentrationinboth A.thaliana andT.salsuginea isgreaterwhenstressedthanwhenunstressed. T.
salsuginea hasahigherbasal levelof prolinethan A.thaliana whenunstressed andhigherlevelsagain
when stressed (Figure 1). Additionally, Figure 1 shows that when unstressed, T. salsuginea
accumulatesalmostthe same concentration of proline asA.thaliana does whensalt-stressed.
4.2. Leaf gDNA and cDNA intron splicing
M 1 2 3 4 5 6 & 7 8 9 10 11 12 13 14 15 16 17 18 19 20
A
C
Figure 2. Agarose gelsof Introns 1-20 inthe leaves ofthe Arabidopsis thaliana P5CS1 gene using Ethidium
bromide (stock solution) tostainthe gel, x0.5 TBE buffer, x6 loading dye (Bioline, UK) andM= the 100 base
pair molecular size marker (Bioline, UK). A = gDNA control, B = cDNA water control and C= cDNA after 3
days of 100 mMNaCl. Gelsviewed under UV light usinga gel-doc system. gDNA and RNAisolated from the
leaves.
1000 bp
500 bp
400 bp
300 bp
200 bp
100 bp
1000 bp
500 bp
400 bp
300 bp
200 bp
100 bp
1000 bp
500 bp
400 bp
300 bp
200 bp
100 bp
B

14
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 predictedspliced 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 differentwithinandbetween bothplantspeciesundercontrol andsalt-stressedconditions.
The gDNA control shown in image A of Figure 2 and image V of Figure 3 verifies there was no
contaminationin the samples.Italsoconfirmed thatall 20intronsare presentintheA.thalianaP5CS1
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 beenoptimisedto the mostsuitable conditionsrequiredforDNA amplificationandgives
the experimenterconfidenceinsubsequentPCRassays. Thisenabledthe successiveanalysisof intron
splicing in both plant species under control and salt-stressed conditions and for a comparison to be
made of betweentheregulationof the P5CS1splicingbothbetweenandwithinthe twoplantspecies.
1000 bp
500 bp
400 bp
300 bp
200 bp
100 bp
1000 bp
500 bp
400 bp
300 bp
200 bp
100 bp
1000 bp
500 bp
400 bp
300 bp
200 bp
100 bp
Figure 3. Agarose gels of Introns 1-19 in the leaves 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). V = gDNA control, W = cDNA water control and Z =
cDNA 3 after days 100 mMNaCl. Gelsviewed under UV light usinga gel-doc system. gDNA andRNA isolated
from the leaves.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
V
W
X

15
Gel image B and C of Figure 2 shows the ampliconsizes(Table1) for all 20 intronsinthe mRNA of the
P5CS1 gene underunstressed(watercontrol) andsubsequentlysalt-stressedconditionsinA.thaliana.
However, no amplicon for intron 2 of A. thaliana under salt-stressed conditions was amplified.
Therefore,noanalysisof the intronsplicingforintron2undersalt-stresscanbe made bothwithinand
betweenthe species. Whencomparingthistoimage Cof Figure 2,there is a cleardifferentialpattern
of intron-mediatedsplicingof the P5CS1gene. Figure2showsthe greatestdifference inintronsplicing
between A. thaliana controls and salt-stressedplants was shown to be between introns 4, 5, 6, 7, 8
and9 as onlyundersalt-stresswasthe splicingof those introns moreoptimal andworkingatfull speed
(image B,C). Figure 2 also showsthatunderbothunstressedandstressedconditions (image B,C) the
splicingof the intronsinthe P5CS1 gene inA.thaliana was neverworkingatfull speed.Nevertheless,
Figure 2 shows intron splicing was enhanced between water control and salt-stressed A. thaliana
(image B,C).
Gel image W andX of Figure 3 shows the amplicon sizes(Table2) forall 19 intronsinthe 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 wasamplified.Therefore,noanalysisof the intronsplicingunderunstressedandsalt-stress
conditions can be made both within and between species. When comparing image W to image X of
Figure 3, there was no difference inthe intron-mediatedsplicingof the P5CS1gene inT. 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 enhancedunder
salt-stressassplicingisalreadyworkingatfull speedincontrol plants(imageW,X).Figure 2(image B)
and Figure 3 (image W) showsthat A. thaliana and T. salsuginea regulate the splicingof theirintrons
differentlyunderunstressed conditionsandthat T. salsuginea hasfewerunsplicedmRNA transcripts.
Figure 2 (image C) andFigure 3 (image X) showsadifferentpatternof intronsplicing undersalt-stress
betweenbothspeciesandthat T. salsuginea hasfewerunsplicedmRNA transcriptsthan A.thaliana.
4.3. Root cDNA intron splicing
Introns5, 6, 7, 8 and 9 were analysedinthe rootsas the optimal splicingandregulation of these
intronswere thoughttoplayan essential role in proline accumulation andsalt-tolerance inP5CS1
genesexpressedinthe leaves. Additionally,rootsplicingwas analysedasthe P5CS1gene isknown

16
to be expressedinthe rootsbutproline does nottoaccumulate there.Thisisbecause prolineis
translocatedtothe leaves.
Figure 4displaysthe patternof intronsplicingof introns5,6,7,8 and9 inthe P5CS1gene inunstressed
and salt-stressedconditionsin A.thaliana andFigure 5 displaysthe splicingof 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
1000 bp
500 bp
400 bp
300 bp
200 bp
100 bp
1000 bp
500 bp
400 bp
300 bp
200 bp
100 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 stainthe 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
viewedunder UV light using a gel-doc system. RNA isolatedfrom the
roots.
M 5 6 & 7 8 9 M 5 6 & 7 8 9
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 stainthe 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
viewedunder UV light using a gel-doc system. RNA isolatedfrom the
roots.
D E
M 5 6 8 9 M 5 6 8 9
Y Z

17
conditionssplicingof intron6and7 wasnot workingatfull speed(Figure 4).Thisagreeswiththe data
shown in image C of Figure 2 and shows that in the roots and leavessplicing 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 splicingof introns5,6, 8 and 9 in the T. salsuginea P5CS1gene tobe enhancedandworking
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 withthe predicted fragment length(Table 2). However, in the
leaves,the lengthof intron8 isapproximately 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,intronsplicinginthe watercontrol (image Y) isnotoptimal (Figure 5). Thisisdifferentto
the resultsshown inimage X of Figure 3, as splicingof 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 hasoptimal splicingof all intronsunlike A.thaliana.
5. Discussion
5.1. Discussionof results
The resultshave confirmedmany of the aimsandhypothesises andprovide somepreliminarydataon
the regulation of the P5CS1 gene in glycophytes and halophytes. Research by eco-physiologistsand
biochemists have shown that proline accumulation is greater in T. salsuginea in comparison to A.
thaliana andthisisvital to itssurvival insalinesoils(Gharsetal.2008). The resultsdisplayedinFigure
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 expressesits P5CS1
gene andthatmechanismsare inplacetoinhibitproline catabolism.Thiscouldbe dueto 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 tosalt-stressand thatthisenablesitssurvival in saline soils. However,itisalsoimportantto
lookat the genome wide responsetosalt-stress.Thisisbecause manyothergenes,suchas PPC1 are
known to be upregulated in response to salt-stress and 75 salt-responsive proteins, such as glycine
betane have beenidentified inT.salsuginea (Changetal.2015). Thissuggeststhat the P5CS1 gene is
part of an extensive,integratedandpreciselymanagedmolecularandphysiological response to salt-
stressin T. salsuginea that still requiresvastresearchtoconfirmthe mechanismsof stress-tolerance.
Figure 2 providesevidence suggestingthatsalt-stressinducesthe expressionof the P5CS1 gene.This
isbecause intronsplicingisenhancedwhencompartingthe control A.thaliana plantstosalt-stressed
(Figure 2). Thismeans that the P5CS1 gene in A. thaliana is differentiallyregulatedundersalt-stress.

18
This suggeststhe gene is expressed undersalt-stressconditionsandmRNA is splicedmore quicklyin
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
responsestosalt-stress(Figure2,3).However,thereisregulationof the P5CS1gene ateverylevel 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
increasedsensitivitytosalt-stressincomparisonto T.salsuginea.
The resultsalsoprovide apossibleexplanationof why T.salsuginea hasbeenshowntocontainhigher
concentrations of proline in both unstressed and salt-stressed plants (Figure 1). Under control and
salt-stressedconditions,thesplicingof the intronsinthe P5CS1geneinthe leavesisdifferentbetween
A.thaliana andT. salsuginea (Figure2,3).However,thereisalsonodifferenceinintronsplicingof 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 working at full
speedbothwhenunstressedandsalt-stressed. Italsosuggeststhat optimal splicinginthe halophyte
may account for its ability to mount an immediate response to salt-stress which is essential to its
survival insaline soils.Thisisphenotypicallyshownbyitssurvival insaline soilsandelevatedproline
levels(Figure 1). Comparingimage C of Figure 2 and image X of Figure 3 shows leaf intronsplicingto
be only fully optimal in T. salsuginea as opposedto A. thaliana. This provides further evidence as to
why T. salsuginea accumulatesmore proline thanA.thaliana undercontrol andsalt-stress(Figure1).
Intron19 is unlikelytobe importantinsalt-toleranceasinbothcontrol andsalt-stressedT.salsuginea
showsemi-optimal splicing(Figure3).
Figure 4 and 5 show root expression and splicing of introns 5, 6, 7, 8 and 9 in the P5CS1 gene in A.
thaliana andintrons5,6, 8 and 9 in T. salsuginea.Figure 4and5 show thatsalt-stressenhances intron
splicing in the roots of both plants. Image D of Figure 4 and image Y of Figure 5 suggest that in the
roots undercontrol conditionssplicingisworking slightlyfasterinA.thaliana. Thiswas 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) and5(imageZ) showthatundersalt-stressintron
splicingwas onlyworkingatfull speedin T. salsuginea.Thiswasexpecteddue topreviousstudies on
salt-tolerance in T. salsuginea and the resultsdisplayed 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 undersalt-stressandthatthisisa vital to its tolerance tohighconcentrationsof NaCl. Itis
also worth noting the importance of spliced fragmentswhensalt-stressed, as optimal splicing under

19
stressis vital tothe resistance shownin T.salsuginea.Thismayexplain whythe presence of unspliced
fragments in the water control of the roots in T. salsuginea does not greatly impact its ability to
accumulate proline. ComparingFigure 2 and4 shows that the splicingof intron6 and 7 inA. 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
phenotypicallyshowing aslowerresponse tosalt-stress.
The resultsforleaf androot P5CS1 intronsplicingshowsthereisadifference insplicingboth 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 subjectedtosalt-stress.Therefore,optimalsplicingundersalt-stressisalikelycomponent
of an efficientresponse tothe stressor.Only T. salsuginea showssplicingtobe workingat full speed
inboththe leavesandthe rootsincontrol(leaves)andsalt-stressed(leavesandroots) conditions.This
poses a new explanation for the salt-tolerant phenotype observed in T. salsuginea. It also highlights
the potential of optimising the regulationof stress-response genesinA.thaliana andsubsequentlyC3
and C4 crops.
5.2. Limitations,critical appraisal and improvementsto the study methods
Potential limitations and criticality include the controversy over the units used to measure proline
accumulation (μmol proline (gFW)−1
).Thisisbecause some molecularbiologistsmayargue 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 taking1 g (FW) of leaf samplesfromboth control and salt-treatedA.thaliana and
T. salsuginea.Therefore,nomisrepresentationaldataof prolineconcentrationinanyof the plantswill
have beenreported.
Additionally,noampliconforsome intronswasachieved andthe timeconstraintof the projectmeant
the optimisationof the primersforthose intronswasnotpossible.A longerperiodof time (24weeks)
to collect the data would have allowed the experimenter to optimise all primers for all introns. This
wouldhave meantthatall intronscouldhave been amplifiedandthatthe splicingof these couldhave
beencomparedbothwithinandbetween species.Thiswouldhave enabledtheanalysisof the splicing
for all 20 intronsin A.thaliana andall 19 in T. salsuginea andmay have shown otherintrons thatplay
a significantrole inthe salt-toleranceobservedin T.salsuginea.

20
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
intronsinthe gene sequence designingindividualprimersforbothintronswasnotpossible.
Due to the time constraintsof the project,onlyintrons5,6,7, 8 and9 of the P5CS1gene 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
allocatedtostudyalternative splicinginbothplants (24 weeks) wouldhave enabledall intronsinthe
roots of both plantsto have beenanalysed.Thiswouldresultedingreaterknowledge of the splicing
of all introns in the roots of both plants under control and salt-stressedconditions and may have
providedfurtherinformationonwhy T. salsuginea isa halophyte.
Additionally, a further improvement could have been to analyse the production of proteins from
unsplicedtranscripts.Thiscouldhave shownwhetherthe unsplicedampliconsshowninFigure 2,3, 4
and 5 were producing any proline biosynthesising enzymes. This would have assisted in the
confromationof prolineaccumulationandhelpedtounderstandanddetermine more accuratelyhow
bothplantspeciesrespondtosalt-stress.
Due to the time constraintsof the project,intronsplicingwasonlyanalysedafter3daysof 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
concentrationsof 100 mM. It is knownthat by day10 the level of the P5CS1 transcript isthe same in
A. thaliana and T. salsuginea but transcriptlevelsplateauatday 3 in T. salsuginea.Investigatingthis
would aid in the understanding of why A. thaliana is slower at preparing its mature transcript.
Additionally,due totime restrictionsonly100 mM of NaCl and itsimpact on intronsplicingbetween
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(Iidaetal.2004) aswell asthe methodsusedinthisstudy.Results would potentially showhow
both plants respond to the initial onset of varying intensities of salt-stress and potentially aid in
confirmingthe characteristicrapidresponseshownin T.salsuginea.

21
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? Isitthe regulationbythe splicesomeorthe differencesinthe intronsequences between
both species (Wu et al. 2012)? Further research should focus on investigating the role of the
splicesomesinthe P5CS1 gene of A.thaliana and T. salsuginea as well asthe differences inbase pair
composition of the intron sequences between both species. This will help to uncover the role the
intronsequences andsplicesomes playinsalt-stress. Thiscouldthenbe appliedtocropspeciesinthe
anticipationof improvingtheregulationof theirstress-responsegenesinordertoincreasecrop yields.
Engineeringthe P5CS1gene of T.salsuginea intoA.thaliana will helptoanswerthese questions. This
is because it would show whether glycophytes have the capacity to regulate the P5CS1 gene in the
same wayhalophytesdo. Therefore,if A.thaliana isunabletoregulatethe P5CS1geneof T.salsuginea
in the same way as T. salsuginea does, this would suggest that the splicesomes are crucial to the
enhancedsplicingandproductionof mRNA seenin T.salsuginea.However,if A.thalianashowsproline
levelsandsplicingsimilarto that of T. salsuginea thenthis wouldsuggestthat it is the differencesin
the intronsequencesbetween A.thaliana andT.salsuginea thatconferits resistance tosalt.
Further research should also focus on the differential impact of other stresseson the splicing of the
introns in both plants. This wouldshow if other introns are differentiallyspliced under other abiotic
stresses such as, drought and heat stress. A comparison of intron splicing of salt, drought and heat
stresscouldthenbe made betweenandwithin unstressedandstressed A.thaliana andT.salsuginea.
This wouldshowthe imact of differentabioticstressesonintronsplicinginbothplants.It may show
splicing of certain introns to be more important to the stress-response of each abiotic stress in both
species. A comparisoncould thenbe made both within and between plant species and a syntheis of
glycophyte andhalophyte differential alternative RNA splicingof the P5CS1gene in A.thaliana andT.
salsuginea underabioticstresscouldbe constructed.
Future workcouldalsoincludeextendingthe analysisbyusingothermethodsof PCRtechnology.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
wouldprovide informationonhowthe expressionof the P5CS1genechangesovertimeinresponseto
salt-stress (Holst-Jensenet al. 2003). Combining this with the data showing intron splicing in both
plants,thiswouldprovide quantitative andqualitative dataonthe abundance of P5CS1 transcriptsin
both A. thaliana and T. salsuginea under control and salt-stressed conditions. The results obtained

22
would help to confirm and validate T. salsuginea as the plant that prepares its mature P5CS1
transcriptsfaster.
Othergenes,suchasthe saltoverlysensitive1(SOS1) have beenshowntobe stronglyinducedbysalt-
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 targetedtoultimatelyenhancethe salt-toleranceof crops andsignifiesthe needtounderstand
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 identifyand ultimately
optimise the regulationof additional genesthe functioninresponsestosalt-stress. Additionally,60%
of regulatedgeneshave beenshowntobe unique to T.salsuginea incomparisonto A.thaliana (Gong
etal.2005). Thissuggeststhatbothplantsrespondextremelydifferentlytosalt-stress. A.thaliana was
showntoemployauniversaldefencepathwaywhereas,T.salsuginea wasshownto upregulategenes
functioning in post-translational modification and protein relocation (Gong et al. 2005). This further
highlightsthe needtounderstandwhole genome responsesandnotjustthe response of one gene to
salt-stress. Future work should focus on bringing together genome responses to salt-stress in both
glycophytesandhalophytes.
The promotorsof the P5CS1gene in A.thaliana andT.salsugineaare slightlydifferent.Furtherstudies
focusing on the promoter between both plants would help to determine if the evolution of salt-
resistance isatthe promoterlevel.Transgenicexperimentsinsertingthe promoterof the P5CS1gene
from T. salsuginea into the P5CS1 gene of A. thaliana and comparing its growth and proline
accumulation toWT A.thaliana insaline soilswouldhelptodetermine 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 shownthose plantstoaccumulate significantlylessproline whensalt-
stressed (Yu et al. 2012). However, the gene is limiting the production of the enzymes and
subsequentlythe biosynthesisof proline.Future workshouldfocuson optimisingthe regul ationand
expression of both the P5CS1 and P5CS2 (duplicatedgene in A.thaliana) genes inorder 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 glycophytesshow to salt-stress,optimistically enabling in the near future the growing and
cultivationof cropsin saline soils.
Kesari etal.(2012) showedproline accumulationtovaryamong A.thaliana strainswhichpresentsthe
possibilityof breedingresistantstrainsof crop plantsto produce more proline.Thiscouldbe carried

23
out using the same methods as Borlaug used in the 20th
century and highlightsthe importance of
retaining and utilising all possible methods to increase crop yields. This would involve screeningfor
varietiesof crops that have higherlevelsof proline accumulationorenhancedefficiencyinleaf/root
splicingof theirP5CS1andotherstress-responsegenes.Crossingtheseplantswouldresultinprogeny
that increased the speed at which they prepare their mRNA and therefore respond more rapidlyto
salt-stress. This could help in selection for A. thaliana and crop ecotypes that do not contain the
harmful exon3skipmutationwhichreducesthe levelof prolineaccumulationandlimitsthe abilityof
glycophytestorespondtosalt-stress(Kesarietal.2012).Thiswouldenhancethe responsecropplants
show to salt-stressif theycontainthisharmful mutation.
Finally, targeted screening of transcription factors, coactivators, histone acetylases and other
potential keysignallingelements,suchasthe protein kinases ORG1may alsoaidin the elucidationof
the mechanismsinvolvedinregulatingthe P5CS1geneandthe generalstress-responses inbothplants
(Nishimura et al. 2005). Understanding the relationship the P5CS1 gene has with its transcription
factors and other signalling elements may reveal differencesbetween the P5CS1 gene in A. thaliana
andT. salsuginea.Thiscouldinturnrevealdifferentmodesof regulatingthe genebetweenbothplant
speciesand may expose the causesof the differencesinthe abilitiesof bothplantstowithstandsalt-
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.Thiswouldresultinhigheryieldswithnogeneticmodificationof the codingsequence. This
would avoid the overall European stigma of GM crops while maximising crop yields and feeding the
world’severgrowingpopulation.
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-
mediatedsplicingof the P5CS1gene tobe preciselyregulated, controlled anddifferentbothbetween
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
glycophytesandhalophytes.
There are three main conclusions to be taken from the results. Firstly, salt-stress inducesthe P5CS1
gene inboth A.thaliana and T. salsuginea.Secondly, Salt-stressenhancesintronsplicinginthe leaves

24
and rootsof A.thaliana andenhancessplicinginthe rootsonlyof T.salsuginea.Thirdly, T.salsuginea
has optimal splicing in the leaves even under unstressed conditions. This advantageously gives T.
salsuginea the capacitytoaccumulate prolinefasterthan A.thaliana.Thisprotects T.salsuginea from
the harmful effectsof salt-stressandenablesitto grow insaline soils. Allthreeconclusionshaveaided
in the molecular and physiological understanding of why T. salsuginea is a halophyte and why it
mountsand immediateresponse tosalt-stress.
However, many questions still remain in regards to the regulation and adaptive value of the P5CS1
gene.Toanswerthese questions extensive investmentin bothcapital andtime isrequiredinorderto
come to a more conclusive culmination of the impact of salt-stress on whole genome regulation in
plants. Furtherresearchisstill neededtobe undertakenbefore the regulationandimportance of the
P5CS1and otherstress-responsegenesare fullyunderstood.Answeringthesequestionswillopen vast
opportunitiesforagricultureandbiotechnology whenaimingatalleviatingthe growingworld biofuel,
feedandmostprominentlyfoodinsecurity.
6. Acknowledgements
Iwouldfirstlyliketoextendmythanksandgratitudetomysupervisor,DrTaharTaybi forthe continual
guidance, support and encouragement he has given me throughout my research. His expertise and
supporthave proventobe vital tomy research.
I wouldlike to give thanksto the laboratorytechniciansinthe School of Biology,Mrs RoselynBrown
and Mrs Miriam Earnshaw. Their support was essential to my overall understanding and successful
completionof laboratorytechniques.
Finally,Iwish to thank Newcastle Universityandinparticular the School of Biologyfor givingme the
opportunitytocarry out thisresearchproject.
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27
8. Appendices
Plant Species gDNA concentration (ng µl-1
) A260/A280
A. thaliana
T. salsuginea
28.00
285.5
2.04
1.86
Plant conditions
Leaf RNA Concentration
(ng µl-1
)
Root RNA Concentration
(ng µl-1
)
A. thaliana T. salsuginea A. thaliana T. salsuginea
Water control
100 mM Nacl for 3 days
334.9
427.9
433.7
534.4
602.0
813.0
963.9
1152.1
Plant conditions
A260/A280 Leaves A260/A280 Roots
Water control
100 mM Nacl for 3 days
2.13
2.14
2.14
2.17
2.09
2.15
2.19
2.17
Table 4. RNA concentrations (ng µl-1) ofextracts from 4-week-old A. thaliana and6-week-old T. salsuginea used
in the RT-PCR. RNA extracted fromthe leaves and roots from water controls andplants 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 ofextracts from 4-week-old A. thaliana and 6-week-oldT. salsuginea usedin
the RT-PCR. RNA samples were read spectrophotometrically at 260/280 nm on the NanoDrop Lite (Thermo
Scientific, UK). A260/A280 values greater than1.8 are suitable for analysis.
Plant conditions
A260/A280
Leaves
A260/A280
Roots
Water control 334.9 433.7 602.0 963.9
100 mMNacl for3 days 427.9 534.4 813.0 1152.1
Table 3. 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).A260/A280 values greater than 1.8 aresuitablefor analysis
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
spectrophotometricallyat 260/280 nm onthe NanoDropLite (ThermoScientific, UK). A260/A280 values greater
than 1.8 are suitable for analysis.

28
(A) Arabidopsis thaliana P5CS1gene sequencetakenfromthe NCBIdatabase. Highlightedinpink is5’
flankingsequence,inyelloware startand stop codons of the CDS, inaqua blue are the exons, ingrey
are the intronsandin red is the 3’ flankingsequences.
CTTCCACGGCGTTTCCTCAGCCGCCGATTTTATTTATTTCCCAAAATACCCATCACCTATAGCGCCACAATCCTCT
ACATCACACCCTAATCTCATTACCATACACCACCCAACGAACACGCGCCACTTCATTTGTTAGTATCTAAAATAC
CAAACCTACCCTTAGTTCCACACGTGGCGTTTCCTGGTTTGATAACAGAGCCTGAGTCTCTGGTGTCGCTGGTG
TTTATAAACCCCTTCATATCTTCCTTGGTGATCTCCACCTTTCCCTCACCTGATATTTATTTTCTTACCTTAAATAC
GACGGTGCTTCACTGAGTCCGACTCAGTTAACTCGTTCCTCTCTCTGTGTGTGGTTTTGGTAGACGACGACGAC
GATAATGGAGGAGCTAGATCGTTCACGTGCTTTTGCCAGAGACGTCAAACGTATCGTCGTTAAGGTTCGTTGA
GATACGTTCGCATTTTCAGATTTTGTTGTTGATGATTAGATTCTTAATTTGTGATAATGTGGAAATGAATATTAT
GTAATTTAAGTGCATCTAAACTCTTTGTTTATTGAATTCGTGAATCTGAATATATTTTCTAATCCCAGAAACTAA
AACTTCTCGTATGAATCTTAATTTGCATGTCATTAGAGACGAATGAATAATCAGAATATTCGAGGGATTTTTTTT
CTGTTTGGTGATTAAAATTTTGGATTTTTGTTTATATTATGTAAAAAAAAAAAGGTTGGGACAGCAGTTGTTAC
TGGAAAAGGTGGAAGATTGGCTCTTGGTCGTTTAGGAGCACTGTGTGAACAGGTAATTGTCAAATTTTAATAA
TCTCCTTTTTGTATTGTGTTTATAAAAAAGTGTAAAGGTTTCATTTTTTTTCACGAAAGACATGTGAAATTATTC
ATGCGTAGTGGCAACTTTAATTTGTAAAAAAATATATATATATAATGTCAGCTTGCGGAATTAAACTCGGATG
GATTTGAGGTGATATTGGTGTCATCTGGTGCGGTTGGTCTTGGCAGGCAAAGGCTTCGTTATCGACAATTAGT
CAATAGCAGGTTAAAGCTTAATGGCTACACTTCATTATTAATCCCTTTCCCTTATAACAACATTTGGAAACAAAA
AAAAAAGGGTGATGATGGATGGACCATTTTGGCTTATGTTTTTATTGCTCAATAACAGTGACATGTGTTTATGT
GTGTTATGATTTAAAAGTTTTGTTTTTTTTTGCTGATGGATTTGTTTTTTTTCTTTTTTTTTGTTAATGGCTTTTGC
AGCTTTGCGGATCTTCAGAAGCCTCAGACTGAACTTGATGGGAAGGCTTGTGCTGGTGTTGGACAAAGCAGT
CTTATGGCTTACTATGAGACTATGTTTGACCAGGTGATTTTTCCTTTGTTATCGAATTCTAGATTATTGTGTAAG
ACATCCAAATATTGATGCTGTTGTTTTTCTTTGGTTAGCTTGATGTGACGGCAGCTCAACTTCTGGTGAATGAC
AGTAGTTTTAGAGACAAGGATTTCAGGAAGCAACTTAATGAAACTGTCAAGTCTATGCTTGATTTGAGGGTTA
TTCCAATTTTCAATGAGAATGATGCTATTAGCACCCGAAGAGCCCCATATCAGGTTTGTCCCTTTTGACATGAA
CTTTTCTACACACTCTGAGATGTGAGGGATTCTTTGAATCTCGTAGTCTAATGTTCAGCTTCACTGGATCTTGAT
ATATGCAGGATTCTTCTGGTATTTTCTGGGATAACGATAGCTTAGCTGCTCTACTGGCGTTGGAACTGAAAGCT
GATCTTCTGATTCTTCTGAGCGATGTTGAAGGTCTTTACACAGGCCCTCCAAGTGATCCTAACTCAAAGTTGAT
CCACACTTTTGTTAAAGAAAAACATCAAGATGAGATTACATTCGGCGACAAATCAAGATTAGGGAGAGGGGG
TATGACTGCAAAAGTCAAAGCTGCAGTCAATGCAGCTTATGCTGGGATTCCTGTCATCATAACCAGGTGAGGA
ACCTTCTAAGCTCACCATGCATAATGATAGGGTGATATGCTTGTTCAAATTTGGTTAGATGGTATATTGATATC
TTTCTTGCTTCTGAAGTGGGTATTCAGCTGAGAACATAGATAAAGTCCTCAGAGGACTACGTGTTGGAACCTT
GTTTCATCAAGATGCTCGTTTATGGGCTCCGATCACAGATTCTAATGCTCGTGACATGGCAGTTGCTGCGAGG
GAAAGTTCCAGAAAGCTTCAGGTAATTGTGACTTATGCATGGCTTTCTTTCATGTTCGTAACGTCAAAAACCAT
TCTTGCTCGGCATAGAGTTACTTAACTTTTTTTTACATTTTGCTATAGGCCTTATCTTCGGAAGACAGGAAAAAA
ATTCTGCTTGATATTGCCGATGCCCTTGAAGCAAATGTTACTACAATCAAAGCTGAGAATGAGTTAGATGTAG
CTTCTGCACAAGAGGCTGGGTTGGAAGAGTCAATGGTGGCTCGCTTAGTTATGACACCTGGAAAGGTAAGAA
AGTATTCATGGCCATAGATAGTTGCTTTTTGTTGCTATGGCTTGGGCAAACATATTGTGCCAATGTAACCTCTC
CTTATTATGTTTCTTATTTTGTGCTTGATAGATCTCGAGCCTTGCAGCTTCAGTTCGTAAGCTAGCTGATATGGA
AGATCCAATCGGCCGTGTTTTAAAGAAAACAGAGGTGATCAGAGGACAATTGTTACCATATAGTTAATTTACA
TACTCTTGAGTTAAATAAGGGATATGACTATCCTCCTAGTTGACATACAATAGTTGTTTATGCTATTTGTTCTTT
GTGGCAATTCCTTTTACAGGTGGCAGATGGTCTTGTCTTAGAGAAGACCTCATCACCATTAGGCGTACTTCTGA
TTGTTTTTGAATCCCGACCTGATGCACTTGTACAGGTATGTTAATAGTCAAAATTCATTTCCCTTCTTAATATGT
GAATTTCCTAAAGCTGTGCTTTATCCACAAACCAAACAGATAGCTTCACTTGCCATCCGTAGTGGAAATGGTCT
TCTGCTGAAGGGTGGAAAGGAGGCCCGGCGATCAAATGCTATCTTACACAAGGTACCATTGCCTCAGATTTCA
TATCATTATTTGCCTCAAAATTTATCACTACAGCTCTTTTAAGTTCATGGTAAATTTCTAGGTGATCACTGATGC
AATTCCAGAGACTGTTGGGGGTAAACTCATTGGACTTGTGACTTCAAGAGAAGAGATTCCTGATTTGCTTAAG

29
GTAAGAACAGATTTACAAGCTAGGAGCTGCAACAGTTCTTTTGTATCTTTTGTTAAACTGGAACCCACCATTTG
CATTTGTGTTACAGCTTGATGACGTTATCGATCTTGTGATCCCAAGAGGAAGCAACAAGCTTGTTACTCAGATA
AAAAATACTACAAAAATCCCTGTGCTAGGTCATGCTGGTATGGTTGCAAGTTTGTTTTTTCCAGAAGATTCTTT
ACTTGGATTGTGCTAGAGTGTGACGATGGCTTAATTGTGTACTTGCAGATGGAATCTGTCATGTATATGTCGA
CAAGGCTTGTGATACGGATATGGCAAAGCGCATAGTTTCTGATGCAAAGTTGGACTATCCAGCAGCCTGTAAT
GCGATGGTAAGAGAACTTTTTACCTTCCATCGAGATTTAATTAATACAGTGGGAGATTCTAAAGTTCAACTGAC
TCATTTCATCTTCTCTCGTCTCTTTCAGGAAACCCTTCTTGTGCATAAGGATCTAGAGCAGAATGCTGTGCTTAA
TGAGCTTATTTTTGCTCTGCAGAGCAATGGTACGTCATAAATGGCCCAATCATTTGTTGGTCTATCTTAACCATT
TATTTGACCTCTTGTTACCTTCCATCTGGATGTCTCATAGATATACATGTAGCCTGTTTGATTATAAATATTGAA
TGGTCATCTCATGAAAACATTTCTAGAGTGGCATAACTCATGAGATATATTAAACTACAGGAGTCACTTTGTAT
GGTGGACCAAGGGCAAGTAAGATACTGAACATACCAGAAGCACGGTCATTCAACCATGAGTACTGTGCCAAG
GCTTGCACTGTTGAAGTTGTAGAAGACGTTTATGGTGCTATAGATCACATTCACCGACATGGGAGGTAGAAAC
TCGACATAACAGGCATTGACTTTAGAAATTCTTTGCATATGTAGTGGAAATGTTCACTCGTTATCTTGTCTTGTA
TGTTGTTACGAGCAGTGCACACACAGACTGCATTGTGACAGAGGATCACGAAGTTGCAGAGCTATTCCTTCGC
CAAGTGGATAGGTAAAGTACTGAATCTTTAACTTGCTTATTATCTGTCTTTGATTTTTCTTGGAAACTGACTGTA
AGATGTTGCGACCTTGAACAGCGCTGCTGTGTTCCACAACGCCAGCACAAGATTCTCAGATGGTTTCCGATTT
GGACTTGGTGCAGAGGTAAGTCAGAGACATACACATAAGTCTATAGATTAAAAACAAATAAAAAGAGGAAGA
GTGAGTGATAAAAAAGTATTGGTTGTGGTATATAGGTGGGGGTAAGCACGGGCAGGATCCATGCTCGTGGTC
CAGTCGGGGTCGAAGGATTACTTACAACGAGATGGTACAATTTTAGTTACTCAAAGCACCATTGTTATGTCAA
TAAAGACCCACAATAAGCCTTTTTTCCTATGCTTCTTTTAATTTTCATGGTGAAATGGTTGCAGGATAATGAGA
GGAAAAGGACAAGTTGTCGACGGAGACAATGGAATTGTTTACACCCATCAGGACATTCCCATCCAAGCTTAAA
CAAGACTTCCGAGTGTGTGTTTGTGTATTTGGTTGAGACTTGAGGAGAGACACAGAGGAGGATGGGCTTTTTT
GTTTCCTCTCTGCTTAGTACTCATATCCTATCATTATTATTATTACTACTACTTATTATTGAAACCCTCGCTTATGT
AGTGGTTTTGATTTAGGGTTAGGATTGCACCAAAAATAAGATCCACTTTACCACTTAGTCTTGCTCATAAGTAC
GATGAAGAACATTTAATTAGCTTCTCTTCTTGTCATTGTAAGCTACCTACACATTTCTGATCTTTATCAAGATACT
ACTACTTTTCATTTCGCTTATCTATAAATATATTTCGATTTGCATTGGAAATCACAAGTTGAATCAGAACTGGAA
ACTCTTAACCATAAATTCTCAAAGATTGTGCTACATTTGAAAGCTAACAATGAACACAAGAAAAGAAC

30
(B) Thellungiella salsuginea P5CS1gene takensequencefromthe Phytozorm database (unpublished).
Highlightedin pink is 5’ flanking sequence, in yellow are the start and stop of the CDS, in aqua blue
are the exons,ingrey are the intronsandinred isthe 3’ flankingsequences.
GACACTTCCCTCACCAGATATTTCCCTAAACGCGCTCACTGACGAAATCCACCACTGAGTTAACTCGTTCCTTCT
CTGGGTTTTGGTAGGCGGCGACAATGGAGGAGCTAGATCGTTCACGCGCTTTTGCCAAAGACGTCAAGCGTA
TCGTCGTTAAGGTCTCGTCTCTCTCTCTCTCTCTCTTTGTATCTGTTTGTTTATCTCCTTATCCGTGTTTCGTTGAG
AAACGTCCGCATTCTCAGATTTTGATTTGATTATCGACTGTTTTTGGCTTAATTGCTGATTTCGATTTTTTTTTGT
TTTTTTTTCTCTGCGTTCGTCTGAATCTGTGAAGTGTTCGTCGTCGTTGGTTGTCGATGTGGATTGGGTTTAGTG
TGTTTTTTAATTTCATTTTAAGCTGTTTTTTGCGGCTGAGTGAAATCTGCGGTAATGTGAAAAATCGAATATTAT
ATGATTTAACGTGCATCTGAATATTTTTTGTTTGTCTCTGTTATTGAAAAGCTCTCAACGGAAAAGTTTCTCGAA
TCTGAATACCATTTGTCTCGGAAAAATTAAACCTCTCGTAATCACGCTTATGAATCTTAATCTGCATGTCATCAG
AGAGTGATGAAGAATCAGAATATTCGGATAATTAATATTCTGTTTTTTTTTTTTTGTAAATATAGGTTGGGACC
GCTGTTGTTACTGGGAAAGGTGGAAGATTGGCTCTTGGTCGCTTAGGAGCACTGTGTGAACAGGTATTTTGAT
TTTTATTATTTACCTTAATTATCATTAACCTATGTTAATTAATCAGCTTTTTGCTTTATTCCTAAATTGTGTAAAAA
GGTTTCACGAAATACATGTGATGCAATTTTGCACCTTTAATTCGTAAAATATATATTATAATGTCAGCTTGCGG
AATTAAACTCGGATGGATTTGAGGTGATTTTGGTGTCATCTGGTGCGGTTGGCCTTGGCAGGCAAAGGCTTCG
ATACAGACAATTAGTCAATAGCAGGTTAAGCAAAATGGCAACTTTTAAACCAATCATTTCACTTTAATCTTATT
GGAATCAAAAAGGGTGATGGACCATTGACTTATGTTTGCTTTCTGATGGGAATAACAGTGAGATGTGTTTATG
ATTTTAAAGTTTTTGTTTTGTGCTGAGTTTATTTCTTAATGGATTGCAGCTTTGCGGATCTTCAGAAGCCTCAGA
GTGAACTTGATGGGAAGGCTTGCGCTGGTGTTGGACAAAGCAGTCTTATGGCTTATTACGAGACTATGTTCGA
CCAGGTGATTTTTCTCTTCTTTTTTTAAGGAAGAAGACTATATATGGTCTCGTTTTCTTAATTGCTGTGTAAAATT
CCAAATATTGATGCTTTGTTTCCTGTTGTTTTCTTTGGTCAGCTGGATGTGACGGCGGCTCAACTTCTGGTGAAT
GACAGTAGTTTTAGAGACAAGGATTTCAGAAAGCAACTTAATGAAACTGTCAAGTCGATGCTTGATTTGAGGG
TTATTCCGATTTTCAATGAGAATGATGCTATAAGCACCAGAAGAGCCCCATATCAGGTTTGTTGACTATCTTTG
GTCCCTTTGAAATGAGTACTCCTTTGAATTTAGCTGCTTCCTATGAATCTCGTAGTCTTATATGTTCAACTTCATT
GCATTTCAATATACGCAGGATTCCTCTGGCATCTTTTGGGATAACGACAGCTTAGCTGCTCTACTGGCGCTGGA
ACTGAAAGCTGACCTTCTGATTCTTCTGAGTGATGTCGAAGGTCTTTACACAGGCCCTCCCAGTGATCCTAACT
CAAAGTTGATCCACACATTTATTAAGGAAAAACATCAAGATGAGATTACATTTGGCGACAAGTCAAGATTAGG
AAGAGGTGGCATGACTGCAAAAGTCAAAGCTGCAGTGAATGCAGCATATGCTGGGATTCCTGTCATCATAAC
CAGGTGTGGGCCCTTTTACATTCATTGTGCATAATTAATACGCTTTCCAAATTTGTCAAGTGTTTTTGATCTCGC
TTTCAGTTCTGACCCTGAATATCATCATCTTAATTCCTCCGAAATACCACAATTTACGTTTGATTGAGAAATATT
CGAAAGATATTTTGTTGGATAGAAAGCTGATACTTTTCTTGCTTTTGAAGTGGGTATTCAGCTGAAAACATAGA
TAAAGTCCTCCGAGGACTGCGTGTTGGAACCTTGTTCCATCAAGATGCTCGTCAATGGGCTCCGATCACAGAT
TCTACTGCTCGTGACATGGCAGTTGCTGCAAGAGAAAGTTCCAGAAAGCTTCAGGTACTGCTAGTTGCTGCAT
GCGTATCTTTTTTCCACAATTATGATGTGAGAAATCTTTTCTTTCTCGGTAGAGATGTATTTAAACTGCTTGTAA
TTTGCTACAGGCCTTATCTTCAGAAGATAGGAAACAAATTCTGTATAATATCGCCGACGCTCTTGAAGCAAATG
AAAAAACAATCAGAGATGAGAATGAATTAGATGTATCTGCAGCACAAGAAGCTGGATTTGAAGAGTCATTGG
TGGCTCGCTTAGTTATGACACCTGCAAAGGTAAGACAGTATTCGTGTTGTGTGGTATTGTGCCAATTTCACCTC
TCCTGATGATCTATATATCTTGTTTTTATTTCTAATGTTTTCTTGTTTTGCTTGATAGATCTCAAGCCTTGCAGCTT
CAGTTCGTAAGCTAGCCGATATGGAAGATCCAATTGGCCGTGTTTTAAAGAAAACTGAGGTGATCAGAGGAC
AGTTGTTATTATATAAAGTTTTACAGTCTAGGAGTATCCTCGTAGTTGACATATAATAGCTGTTTATCCTATTCG
TTCATCGTGACAATTGCTTTTACAGGTGGCAGATGGTCTTGTCTTAGAGAAGACCTCATCCCCATTAGGCGTAC
TCCTGATTGTTTTTGAATCCCGACCTGATGCACTTGTACAGGTATGTTAAGAGTCAATGTCCTTTTATCTTCTTA
GAATGTGAATTTGCTGAAACCTGTGTTTTATCCACAAACCAAACAGATAGCTTCACTTGCCATCCGGAGTGGA
AATGGTCTTCTATTGAAGGGTGGAAAGGAGGCCCGGCGATCAAATGCTATCTTACATAAGGTACAGTGCCTCA
GATTTCAGACTCGGATGTTATCATATATGGCTTCCTCAAAATATGCTGGTTATAATTGATCCATTTAATTTCATT
TTAAATTTCTAGGTGATCACTGATGCAATTCCAGAGACTGTCGGGGGTAAACTCATTGGACTTGTGACTTCAA

31
GAGAAGAGATTCCTGATTTGCTCAAGGTAAACGGATTTACAAACTTGGAGCTGCAACAAATCTTTATATCTTGT
GTTTAAATGGAAACCACCATTTGCATTTGCGCTACAGCTTGATGACGTTATAGATCTTGTGATCCCAAGAGGCA
GCAACAAGCTTGTTTCCCAGATAAAAAATACTACAAAAATCCCTGTGCTAGGCCATGCTGGTACGGTTTCAAG
TTTGTTTTTCCATAAAATTCTTTAGTTGGATTGTGTTAGAGAGTGACTGTCTTAATTTTGTACTTCCAGATGGAA
TCTGTCATGTATATGTCGACAAGTCATGTAATCTGGATATGGCAAAGCGCATAATTTCCGATGCAAAGTTGGA
TTATCCAGCAGCCTGTAATGCGATGGTAAGAGAACTTGTACCAGCCTCTTGAGATTGGAGTATGCAATGGGCG
TATTAATTTCATCCGACTCATTTCACCTTCTCTTTCCTTTGTATTTTCAGGAAACTCTTCTTGTGCATAAGGATCT
AGAGCAGAACGGGCTCAATGAGCTTATTTTTGTGCTGCAGAGCAATGGTATGTCATAAATGCCGTGTTTGTTG
GTCTCTCGTAATCCTGAAGACTTTTTTTTTGGTTGGTAAAATTAATTCTGAAGACTTGTTTGGAGTAATTTAACT
CATGAAGTATTTTTAACTGCAGGAGTCACTGTATATGGTGGACCAAGAGCAAGTGCAATACTGAACATACCAG
AAGCACGGTCGTTCAACTATGAGTACTGTTCCAAGGCTTGCACCGTTGAAGTTGTAGAAGACGTTTACGGTGC
TATAGATCACATTCACCGACATGGGAGGTAAAAACTCGATATAACAGACATTGAGTTTTGTAATCTTTTTGCCT
ATGTACTGGAAATGTTCACTCTTTATCTTGTCTTATATTTTGTTACGAGCAGTGCGCACACAGATTGCATTGTGA
CAGAGGATACCGAAGTTGCAGAGCTATTCCTTCGCCAAGTGGACAGGTAAAATACCGGATCATGAACTTGTTT
AGTGGCTGTCTTTGATTATGTTGGTAACTGACTGTAAGATGTACGTCCTTGAACAGCGCTGCTGTTTTCCACAA
CGCAAGCACAAGATTCTCAGATGGGGCTCGATTTGGACTTGGTGCCGAGGTAAGTGAGAGACATACAAATAA
TCCTATTTATCAAACAGGGAAAAGAGGGAAGAGTGAGTGATGAAGTAAGTTTTGGTTGGTTATACATAGGTG
GGAATAAGCACAGGTAGGATTCATGCTCGTGGCCCAGTCGGAGTTGAAGGATTACTTACAACAAGATGGTAC
CATTTTACTTACTTGAAACACCATTGTTGTTATGTCGATATATCCTCGCAATAAGCTTTTTCTTCTTAGCTTTATTT
GTAAATTTTCATGGTGAAATGGTTTGAAGTATGAGTGATGGTGGTTGCAGGTTAATGAGAGGAAAAGGACAA
GTTGTTGATGGAGACAATGGGATTGCTTACACCCATCAAGACATTCCCATCCAATCTTAGAAGACTGTTGTGTG
TTGAGACTTGAGGAGAGGATGGGCTTTTTGTTTCCTCTCTGCTAATATCATATCCTATTATTATTGTTATTGAAA
CCCTCTCTTATGTAGTGGTTTTGATTTAGGAATTAGGGATTGCACCAAGAATAAGTTACCACTTGGTCTTGCTC
ATAAGTAAGATGAAGAACATTTTCTTAGCTTCTCTTCTTGTTTAAAAAAAACACGTTGTAAGGCTACCTACACCT
TTCTGATTTATCATTTATCTATATCTTTGGATTTGAGTTTGGACTTCCACTGGGAGTTATACCTTTAATACAAAGT
TGCATATATGAACTTAAAAAGTCATTACTATTAATTCCCAAGGATCAGCGCAAAATGGTAAACACGTTTGAGTA
CGTTTGAGTGCTAAGAATGAACACAAGAGTTCTCATTCTTACATATCTAATTTTTTTCAGGTACTTGAGGAATC
GATCTTAACTTTCATTCTTCCATAAACTTCTTAACATTCTTCAACCACAGCAAGTACTCTCGCTTCACTTTTCTCAT
CATGTACTZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZ

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