Yeast functional screen to identify genetic determinants capable of conferring abiotic stress ...

15 Pages·2010·5.34 MB·English

Eswaranetal.BMCBiotechnology2010,10:23
http://www.biomedcentral.com/1472-6750/10/23
RESEARCH ARTICLE Open Access
Yeast functional screen to identify genetic
determinants capable of conferring abiotic stress
tolerance in Jatropha curcas
Nalini Eswaran1, Sriram Parameswaran1,2, Balaji Sathram1, Bhagyam Anantharaman1, Raja Krishna Kumar G1,
Sudhakar Johnson Tangirala1*
Abstract
Background: Environmentally inflicted stresses such as salinity and drought limit the plant productivity both in
natural and agricultural system. Increasing emphasis has been directed to molecular breeding strategies to
enhance the intrinsic ability of plant to survive stress conditions. Functional screens in microorganisms with
heterologous genes are a rapid, effective and powerful tool to identify stress tolerant genes in plants. Jatropha
curcas (Physic nut) has been identified as a potential source of biodiesel plant. In order to improve its productivity
under stress conditions to benefit commercial plantations, we initiated prospecting of novel genes expressed
during stress in J. curcas that can be utilized to enhance stress tolerance ability of plant.
Results: To identify genes expressed during salt tolerance, cDNA expression libraries were constructed from salt-
stressed roots of J. curcas, regulated under the control of the yeast GAL1 system. Using a replica based screening,
twenty thousand yeast transformants were screened to identify transformants expressing heterologous gene
sequences from J. curcas with enhanced ability to tolerate stress. From the screen we obtained 32 full length
genes from J. curcas [GenBank accession numbers FJ489601-FJ489611, FJ619041-FJ619057 and FJ623457-FJ623460]
that can confer abiotic stress tolerance. As a part of this screen, we optimized conditions for salt stress in J. curcas,
defined parameters for salt stress in yeast, as well as isolated three salt hypersensitive yeast strains shs-2, shs-6 and
shs-8 generated through a process of random mutagenesis, and exhibited growth retardation beyond 750 mM
NaCl. Further, we demonstrated complementation of the salt sensitive phenotypes in the shs mutants, and
analyzed the expression patterns for selected J. curcas genes obtained from the screen in both leaf and root
tissues after salt stress treatments.
Conclusions: The approach described in this report provides a rapid and universal assay system for large scale
screening of genes for varied abiotic stress tolerance within a short span of time. Using this screening strategy we
could isolate both genes with previously known function in stress tolerance as well as novel sequences with yet
unknown function in salt stress tolerance from J. curcas. The isolated genes could be over-expressed using plant
expression system to generate and evaluate transgenic plants for stress tolerance as well as be used as markers for
breeding salt stress tolerance in plants.
Background plant productivity. According to Bayer [1], abiotic stres-
Environmentally inflicted stresses such as extreme tem- ses are estimated to reduce yields to less than half of
peratures, low water availability, high salt levels, mineral that possible under ideal growing conditions. The efforts
deficiency and toxicity are frequently encountered by to improve crop performance under environmental
plants both in natural and agricultural system that affect stresses have been moderately successful as the funda-
mental mechanisms of stress tolerance in plants are yet
*Correspondence:[email protected] to be completely understood. Conventional approaches
1PlantMetabolicEngineeringGroup,RelianceLifeSciencesPvtLtd, to breeding crop plants with improved stress tolerance
DhirubhaiAmbaniLifeSciencesCenter,R-282,Thane-BelapurRoad,Rabale,
have thus far met with limited success because of the
NaviMumbai-400701,India
©2010Eswaranetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
Eswaranetal.BMCBiotechnology2010,10:23 Page2of15
http://www.biomedcentral.com/1472-6750/10/23
difficulty of breeding tolerance associated traits from Jatropha curcas (Physic nut), a member of Euphorbia-
diverse plant backgrounds. Hence, an increasing empha- ceae family has been identified as a biodiesel plant in
sis has been directed to molecular strategies targeted at tropical countries [12]. Being a Euphorbiaceae member,
enhancing the intrinsic ability of the plants to survive J. curcas, is to a certain extent, drought tolerant and has
stress conditions. Current approaches proposed to date been documented in certain instances to grow in heavy
focus attention on identification of genes associated metal contaminated soils [13]. Commercial plantation of
with salinity, drought and abiotic stress resistance, fol- J. curcas has been proposed as a sustainable source of
lowed by genetic modification of the plants expressing biodiesel mainly due to high seed oil content, lipid com-
genes enabling them to withstand restrictive growth position similar to that of fossil diesel and non-compet-
imposed by unfavourable environmental conditions [2]. ing demand with edible oil supplies [14]. Prospecting of
Functional screening of microorganisms that express novel genes expressed during stress tolerance from
heterologous cDNA libraries is a powerful tool for iden- J. curcas and utilizing genetic information to modify and
tifying genes with specific functions, independent of the enhance the stress tolerance ability of this plant may
regulation of their expression [3]. The screening of enable development of J. curcas lines capable of produ-
E. coli or yeast expressing plant cDNAs has been used cing growth in poor and marginal soils with acceptable
successfully to identify genes that are involved in oilyields.Besides,thesenovelgenesisolatedfromJ.curcas
enhanced stress tolerance [4]. Functional screening of may also be applied for genetic improvement of other
sodium-sensitive yeast expressing a cDNA library of the agriculturallyimportantcropsmediatedviaheterologous
halotolerant plant sugar beet resulted in identification of geneexpressionortodevelopsalineanddroughttolerant
the eukaryotic translation initiation factor (eIF1A), and varieties.
overexpression of the eIF1A has been reported to However, due to limited genome information, large
increase salt tolerance of yeast and Arabidopsis [5]. Cad- scale gene prospecting to identify genes that are linked
mium (II)-sensitive yeast mutant ycf1 has been to resistance to abiotic stress in J. curcas has been chal-
employed to screen Arabidopsis cDNA library to iden- lenging. To overcome this limitation, we describe a
tify cadmium resistance gene, AtPcrs [6]. The genes for functional genetic screen that utilizes yeast as a surro-
allene oxide cyclase and the cytosolic chaperonin con- gate system to identify and isolate genes involved in
taining TCP-1a homologue [4] have been isolated as salt stress tolerance. We have used yeast GAL1 regulated,
tolerant genes from B. sexangula by functional screening cDNA expression libraries derived from J. curcas, to
on NaCl-containing medium using E. coli as the host develop replica printing based functional screen for
organism. More recently, Ezawa and Tada [4] reported identification of yeast transformants expressing heterolo-
functional screening in Agrobacterium as an effective gous genes that confer abiotic stress tolerance. Through
supplemental method to pre-screen genes involved in the above screen, we have demonstrated the ability to
abiotic stress tolerance. These results demonstrate that isolate several novel genes specific to J. curcas expressed
microbial functional screening is an effective tool to during abiotic stress tolerance.
quickly identify stress tolerant gene candidates in plants.
Genetic engineering and modern molecular breeding Results and discussion
methods have been used to isolate key genes involved in Identification of salt stress condition for wild-type yeasts
abiotic stress response from characterized model plants and isolation of salt hypersensitive yeast mutants, shs-2,
such as Arabidopsis, and several economically important shs-6 and shs-8
crops [7,8]. Directly applying these strategies to non- To identify and isolate J. curcas genes involved in salt
model plants is often challenging. Several other tolerance, yeast cells were used for functional screening.
approaches such as DD-RT-PCR, subtractive hybridiza- Yeast cells have been shown to exhibit salt stress, which
tion, microarray analysis and FOX screening have also is reflected as retardation in cell growth. Wild type yeast
led to identification of genes regulated during stress strains have been reported to show salt sensitivity from
response [2,9,10]. However, a functional effect of the 500 mM depending on growth conditions. In our assays
gene(s) identified on stress resistance is not always we scored for survival of yeast-transformants that could
clearly established from such experimental approaches survive at and beyond 750 mM NaCl. This condition
[11]. While it has been possible in certain instances to selected for the screen at a stringent concentration of
extend and utilize the information available on genes 750 mM NaCl, subjected the yeast transformants to
regulating and/or conferring salinity, drought and abio- ionic as well as osmotic stress, and imposed stringent
tic stress resistance in model plants, the ability to conditions for salt and drought stresses. Further, to
uncover novel or target plant specific genes/pathways enhance sensitivity of the screen, three salt hypersensi-
regulating abiotic stress tolerance would be limited tive mutants shs-2, shs-6 and shs-8 were generated by
without a de novo screen. random mutagenesis that exhibited growth retardation
Eswaranetal.BMCBiotechnology2010,10:23 Page3of15
http://www.biomedcentral.com/1472-6750/10/23
Figure1Identificationofsaltstressconditionsforwild-typeS.cerevisiae(BY4741).Sectorsinplatesmarkedwithreddotarewild-type,
whileothersectorsrepresentsalthypersensitiveyeastmutantsobtainedthroughrandomUVmutagenesis.Wild-typeyeastBY4741showssalt
sensitivityfrom500mMNaCl,withcompletegrowtharrestat2.0MNaCl.Incontrast,salthypersensitivemutantsshs-2,shs-4,shs-6andshs-8
(isolatedintheBY4741background)showgrowthretardationfrom500mM.Similaramountofinoculumswereusedinalltheplates.
beyond 500 mM NaCl (Figure 1). The salt hypersensitive or when the seedlings were treated for prolonged time
mutant shs-2 was used to demonstrate the ability of (see additional file 1). Further, from this time-course
recovered J. curcas genes to complement the salt sensi- analysis, for salinity stress phenotype in J. curcas it was
tive defect in this mutant. identified that the exposure of roots to 150 mM NaCl
The underlying basis for the screen is the ability to for 2 h, at a relative humidity of 40-50% was sufficient
use a catabolic regulated GAL1 promoter [15] to condi- to observe early effects of salt stress, which was marked
tionally express a J. curcas gene in the yeast cells, cloned by low level-wilting of young leaf tissue prior to stem
into a yeast shuttle vector [16]; while scoring the relative drooping (see additional file 1: salinity treatment to
survival of yeast cells exposed to salinity or other abiotic seedlings).
stress to the same yeast transformants, grown under Salt and stress pathways in plants are perceived by sig-
repressed state, subject to similar stress or under control nalling networks and regulated by transcription machin-
conditions. Scoring the relative growth patterns allows ery [17]. To enrich cDNA libraries for transcripts that
the identification and characterization of the tolerant represent a greater proportion of early acting factor to
transformants by plasmid-rescue (Figure 2). later responding regulators, we sampled root tissue at
the early-time points of salt stress. J. curcas root tissue
Construction of cDNA library from J. curcas to identify samples treated at 150 mM, for 2 h time point were
stress responsive genes used in construction of cDNA libraries using the
To understand the early salt-stress phenotypes and char- SMART cDNA synthesis Kit [18] (see Methods).
acterize the genes involved in early responses to salt While plant genomes display large size variations, the
stress on J. curcas, 3-4 week old seedlings were treated number of expressed complement of genes involved in
with various levels of salt stress (see additional file 1: cellular processes are more conserved. Based on the
salinity treatment to seedlings). The phenotypic effects information available for model plant Arabidopsis and
of salt stress resulted in observable wilting and drooping rice, ~3000-5000 genes out of a transcriptome of
in J. curcas seedlings. The effect of salt stress was evi- ~27000 genes are expressed in root tissue [19,20].
dent at a concentration of 150 mM NaCl, and the Recent analysis indicates a modest genome size for the
effects were pronounced at higher salt concentrations, J. curcas, among Euphorbiaceae members, with an
Eswaranetal.BMCBiotechnology2010,10:23 Page4of15
http://www.biomedcentral.com/1472-6750/10/23
Figure2Schematicillustrationoftheunderlyingprincipleofthefunctionalgeneticscreen.IdentificationandisolationofJ.curcasgenes
conferringresistancetoabioticstressinS.cerevisiae.Inpanel(A)growthofS.cerevisiaecoloniesunderabioticstressconditionsacquireddueto
heterologousgeneexpressionwasrepresented.Inpanel(B)transformationandscreeningofJ.curcasrootcDNAlibraryinS.cerevisiaeforability
tosurvivestressusinginduciblepromoterwasrepresented.
estimated genome size of ~450 Mb that provide an criteria included scoring relative survival advantage, for
approximation of transcriptome complexity [21]. To each transformant when subject to stress and gene
adequately represent root transcriptome, titers repre- expression status (Figures 2 and 3). This method
senting ~48000 c.f.u were recovered in each of the un- increased efficiency of gene recovery and reduced the
amplified pools that largely comprised of full-length screening errors by providing additional, more exhaus-
cDNA sequences in 45-50% of the genes. This library tive controls that allowed the identification and elimina-
was used for subsequent functional analysis in yeast. tion of both false-positive as well as false-negative
phenotypes. The screening conditions select yeast trans-
Yeast based replica-printing method for isolating J. curcas formants that are capable of survival under stress
genes involved in abiotic stress (acquired due to expression of J. curcas heterologous
To increase the probability of representation of each cDNA) and are able to grow; while eliminating events
individual clone within these yeast expression libraries due to lethality, non-expression and other experimental
and to efficiently transform yeast, we amplified the copy artefacts.
numbers of clones contained in these libraries in E. coli Using the replica-printing based functional screen, 345
(see Methods for details). A total of 20,000 yeast trans- candidate yeast transformants expressing cDNA (derived
formants (for the plasmid borne URA3 marker) repre- from J. curcas roots) were isolated from a pool of 20,000
senting the J. curcas root cDNA library was screened for yeast transformants. A representative data set showing
salinity tolerance using a replica printing method (Figure results of phenotypic selection of salt tolerance is pre-
3). The replica printing process was devised to identify sented in (Figure 4). The transformed yeast cells dis-
and isolate yeast transformants expressing heterologous played an enhanced ability to tolerate unfavourable
gene sequences derived from J. curcas root cDNA conditions imposed by salinity stress at a stringent selec-
libraries, with a low-rate of false negatives. Screening tion of 750 mM NaCl concentration.
Eswaranetal.BMCBiotechnology2010,10:23 Page5of15
http://www.biomedcentral.com/1472-6750/10/23
Figure3Replicaprinting.Schematicoutlineofthequadrupletplatebasedreplica-printingtoscreenyeasttransformantstosaltstress.Inthis
assayeachindividualyeasttransformantisplacedonfourselectiveplates.Comparisonofgrowthofyeastbetweentheseplatesallowsscoring
andisolationoftransformantsexpressinggenes,conferringstresstolerance.
Plasmid rescue, complementation of salt sensitive yeast demonstrated the ability of the plasmid encoded genes
phenotypes and sequence analysis derived from J. curcas permitted survival of wild-type
To determine and characterize J. curcas cDNA gene yeast transformants (data not shown), and complemen-
sequences that conferred enhanced salinity tolerance at ted several salt sensitive phenotypes of the shs-2
750 mM NaCl to yeast transformants plasmid rescue mutants. The data provided functional support for the
was performed as described in Bates [22]. Analysis of consistent ability of the recovered genes to confer salt
the DNA sequence information, recovered from the tolerance, and support cell growth at conditions other-
yeast transformants showed that 32 sequences from 345 wise detrimental to cell survival.
clones encoded full-length genes with assignable ORF’s
derived from J. curcas; while the remaining were trun- Computational sequence, annotation and analysis reveals
cated cDNA sequences. The ability to recover genes novel abiotic stress tolerant genes from J. curcas
from J. curcas involved in these processes demonstrates Further, sequence analysis and computational annota-
the efficacy of our strategy. tion reveals presence of novel genes from J. curcas
To demonstrate the robustness of the screening involved in abiotic stress tolerance (Table 1). To anno-
method, we have re-transformed the rescued plasmids tate the sequences, computational searches were per-
into wild-type yeast (data not shown) and salt hypersen- formed for similarity to sequences deposited in plant
sitive mutant shs-2 (Figure 5). We evaluated these trans- sequence databases (see Methods), that suggest the pre-
formants for growth at high-salt stress of 750 mM or sence of plant specific regions within these sequences
higher concentration. Several transformants of shs-2 (Table 1). In this analysis 31 out of 32 (~96%) sequences
mutant allowed growth at 750 mM NaCl. The data with assignable hit to the sequence databases showed
Eswaranetal.BMCBiotechnology2010,10:23 Page6of15
http://www.biomedcentral.com/1472-6750/10/23
Figure4YeasttransformantsexpressinggenesderivedfromJ.curcasrootlibrary.Photographsofrepresentativereplicaplatescreen(as
describedinFigure3)demonstratingtheabilitytoisolateyeasttransformantsduringsalinitystress.Yeasttransformants(selectedforplasmid
borneURA3marker),expressinggenesderivedfromJ.curcasrootlibraries,showingtolerancetostressinducedby750mMNaClaremarkedin
panelD(blackcircles).Identicalyeasttransformants,(comparepanelBtoD)withrepressedgeneexpressionshowsarrestedgrowth.(A)
describescontrolconditionswheregeneisrepressedandstressisnotprovidedtotheyeastcells.Yeasttransformantsareexpectedtogrowon
syntheticselectionplateswithdextroseasthecarbonsource.Transformantsintype(B)conditions(toidentifyfalse-positives)aregrownin
syntheticselectionmediawithdextroseasthecarbonsource,buttreatedunderstressconditions.Intheseplatesalltransformantsareexpected
toshowretardedgrowth,duetothestressconditions.Intype(C)conditions,yeasttransformantsaregrownonsyntheticselectionplates
containinggalactose,butwithoutsubjectingthemtostressconditions.Iftheexpressionofanyheterologousgeneisdetrimentaltocellgrown,
itcanbeidentified,thuseliminatingrecoveryoffalse-negativetransformantsinthescreen.Intype(D)conditions,wherethegeneexpressionis
inducedbygalactoseandthecellsaresimultaneouslysubjectedtostress,yeasttransformantsthatarecapableofsurvivalunderstress(acquired
duetoexpressionofJ.curcasheterologouscDNA)areabletogrow(Figure3).
matches greater than the accepted threshold as elabo- role in conferring abiotic stress tolerance in other plant
rated by Altshul et al. [23] to other known plant genes; species (Table 1).
while for a single sequence, significant matches to exist- To understand the possible role of the genes and reg-
ing sequences in the sequence database was not appar- ulatory pathway for abiotic stress tolerance in J. curcas,
ent (Table 1). This sequence could represent novel we assigned functional classes for the gene sequences
determinant of salinity and drought stress tolerance, recovered in the present study. Comparison of func-
whose role needs to be further established. Of the 31 tional annotation for several of the sequences obtained
sequences, 7 sequences showed match to encoded ribo- from plasmid rescue indicated a possible role in toler-
somal proteins, while 1 sequence was annotated as a ance to abiotic stress. Some of the sequence orthologs
eukaryotic translation initiation factor SUI1. Twenty of these genes have been reported to be involved in con-
three sequences have been reported to have a strong ferring stress tolerance in other plant species. We find
Eswaranetal.BMCBiotechnology2010,10:23 Page7of15
http://www.biomedcentral.com/1472-6750/10/23
Figure5Functionalevaluationoftransformantsinsalthypersensitive(shs-2)mutant.Plasmidtransformationofshs-2withfiveselected
genes.235,lateembryogenesisabundantprotein-5;392,cytosolicascorbateperoxidase-1;63,metallothionein;513,mitochondrialATPsynthase
6KDsubunit;619,profilin.SinglecolonyofeachtransformantsandmutantstrainwasplatedonYPDmediacontainingrangeofNaCl
concentrations(0mM,250mM,500mM,750mMand1M).
among the list of known functions sequence classes as cold,drought,UVlight,salinityandwounding[29].In
mechanistically involved in abiotic stress tolerance due this report, we isolated J. curcas LEA-5 like sequences,
to wounding, response to cellular redox or oxidative suggesting a conservation in stress response pathway.
damage, and modulation of cell physiology in response Additionally,weidentifiedgenesencodingmajorallergen
to stresses. For example, previous investigations indicate Pru ar1-like proteins, defensin and protease inhibitor/
the involvement of metallothioneins [24], thioredoxins, seedstorage/lipidtransferproteinfamily reportedtoget
cellular redox machinery [25,26] and factors involved in up-regulateduponpathogenattackand/orenvironmental
the regulation of protein translation [27] to be asso- stimuli [30]. Plants have been known to have specific
ciated with stress resistance in rice and Arabidopsis. mechanism for detoxification of reactive oxygen species
Among gene sequences listed in Table 1, the most nota- (ROS)induced in stressresponse which includesactiva-
ble examples with implied roles in stress tolerance are tionexpressionofantioxidant enzymessuchascytosolic
discussed below: we isolated a sequence ortholog corre- ascorbate peroxidase-1 (Apx1). Experiments involving
sponding to allene oxide cyclase. Sequences of allene over-expression of Apx-1 in transgenic tobacco, con-
oxide cyclase, isolated from mangrove have been ferred enhanced tolerance to salt and water stress, by
reported to confer salinity stress tolerance in multiple effectively detoxifying accumulated H O in the chloro-
2 2
organisms [28]. Previous research data also indicate that plasts [31]. Mitochondrial ATP synthase 6 KD subunit
accumulation of late embryogenesis abundant protein (MtATP6)isreportedtobeinvolvedinNaHCO induced
3
(LEA-5) transcripts in response to stress conditions such stress, whose over-expression in tobacco led to greater
Eswaranetal.BMCBiotechnology2010,10:23 Page8of15
http://www.biomedcentral.com/1472-6750/10/23
Table 1Annotation of32full lengthgenes, derived fromJ. curcas,obtained from yeast functional genetic screen
tolerant to abioticstress*
Sr. CloneID GenBank Putativefunctionofgene Lengthofgene References*
No. AccessionNo. (bp)
1. JcRHDYT30 FJ489601 Alleneoxidecyclase 777 Yamadaetal.2002[28]
2. JcRHDYT70 FJ489602 ThioredoxinH-type(TRX-h) 357 Hongetal.2004[25];Serratoetal.
2004[26]
3. JcRHDYT63 FJ489603 Metallothionein 234 Jinetal.2006[24]
4. JcRHDYT49 FJ489604 Heterotrophicferredoxin 492 SheokandandBrewin,2003[41]
5. JcRHDYT91 FJ489605 Defensin 234 Zhangetal.2008[42]
6. JcRHDYT95 FJ489606 Calmodulin-7(CAM-7) 810 Pardoetal.1998[43]
7. JcRHDYT29 FJ489607 MajorallergenPruar1-likeprotein 495 Mbeguieetal.1997[30]
8. JcRHDYT19 FJ489608 S18.Aribosomalprotein 495 Williamsetal.2003[27]
9. JcRHDYT69 FJ489609 60SribosomalproteinL18a 537 Carrolletal.2008[44]
10. JcRHDYT108 FJ489610 Proteaseinhibitor/seedstorage/lipidtransfer 348 Choietal.2008[45]
proteinfamily
11. JcRHDYT174 FJ489611 Unknownprotein 195
12. JcRHDYT231 FJ619041 Membraneprotein-2 189
13. JcRHDYT235 FJ619042 Lateembryogenesisabundantprotein5(LEA-5) 267 HundertmarkandHincha,2008
[29]
14. JcRHDYT365 FJ619043 Cold-inducedplasmamembraneprotein 174 Imaietal.2005[46]
15. JcRHDYT392 FJ619044 Cytosolicascorbateperoxidase-1(Apx-1) 753 Badawietal.2004[31]
16. JcRHDYT619 FJ619045 Profilin-likeprotein 384 Ramachandranetal.2000[33]
17. JcRHDYT618 FJ619046 Caffeoyl-CoA-O-methyltransferase(CCoAOMT) 741 Dayetal.2009[47]
18. JcRHDYT557 FJ619047 EukaryotictranslationinitiationfactorSUI1 381 Langlandetal.1996[48]
19. JcRHDYT401 FJ619048 Copperchaperone 282 WintzandVulpe,2002[32]
20. JcRHDYT391 FJ619049 Ubiquitinconjugatingenzyme2(JcE2) 447 Criquietal.2002[49]
21. JcRHDYT513 FJ619050 MitochondrialATPsynthase6KDsubunit 171 Zhangetal.2006[17]
(JcMtATP6)
22. JcRHDYT442 FJ619051 Ferritin-2,chloroplastprecursor 771 Masudaetal.2001[50]
23. JcRHDYT483 FJ619052 Annexin-likeprotein 945 Gidroletal.1996[51]
24. JcRHDYTC-4 FJ619053 Al-inducedprotein 711 Tayloretal.1997[52]
25. JcRHDYT399 FJ619054 Avr9/cf-9rapidlyelicited(JcACRE)gene 231 Rowlandetal.2005[53]
26. JcRHDYT354 FJ619055 60SribosomalproteinL39 156 Carrolletal.2008[44]
27. JcRHDYT370 FJ619056 RibosomalproteinL37 291 Carrolletal.2008[44]
28. JcRHDYT381 FJ619057 RibosomalproteinL15 729 Carrolletal.2008[44]
29. JcRHDYT413 FJ623457 40SribosomalproteinS15 456 Carrolletal.2008[44]
30. JcRHDYT461 FJ623458 40SribosomalS18 459 VanLijsebetkensetal.1994[54]
31. JcRHDYT512 FJ623459 Plantlipidtransfer/seedstorage/trypsin-alpha 306 Linetal.2005[55]
amylaseinhibitor
32. JcRHDYT591 FJ623460 Low-molecularweightcysteinerich69 234 Vanoosthuyseetal.2001[56]
*Sequenceorthologwithpreviousreportstoabioticstresstolerancehavebeenmarkedwithreferencesinthetable.
saltstresstolerance [17].Al-inducedprotein andcopper of occurrence of functionally conserved genetic compo-
chaperonehavebeenimplicatedtoprovideprotectionto nents indicates a possible conservation in some of the
cellular function during cytotoxic reactions caused by geneticpathwaysand/orthegenesregulatingsalinityand
metalions[32].WeidentifiedJ.curcassequenceortholog drought stress among diverse plants [2]. The fact that
ofaprofilin.Profilinshavepreviouslybeen implicatedin someofthesefactorsareconservedsuggeststhatcertain
root elongation and root hair system, a response that is aspectsofmechanismofsaltandwaterdeficitstresssen-
directly linkedtoresponse ofplantstoadaptandsource sing and response may be evolutionarily conserved
waterduringdroughtresistance[33].Thehighfrequency amongdiversetaxa.
Eswaranetal.BMCBiotechnology2010,10:23 Page9of15
http://www.biomedcentral.com/1472-6750/10/23
Gene-expression analysis of selected J. curcas sequences varied modes of gene-regulation between the leaf and
involved in salinity stress tolerance using semi- root tissue (Compare Figure 6 to Figure 7). The data
quantitative RT-PCR indicates a complex framework for gene regulation dur-
To understand and correlate the gene expression pattern ing adaptation to salt stress in different tissues. The data
of selected genes to functional roles in stress determina- in conjunction with gene function may help unravel cer-
tion, we performed gene expression analysis in leaf and tain aspects of plant abiotic stress response.
root tissues of J. curcas. In the present study, we
selected five genes i.e., late embryogenesis abundant pro- Conclusion
tein 5 (LEA-5), cytosolic ascorbate peroxidase (Apx-1), The approach described in the report provides a rapid
metallothionein, profilin and annexin, based on the and universal assay system for large scale screening of
diverse functional classes and varied mechanismsimpli- genes for varied abiotic stress tolerance within a short
cated in stress avoidance/tolerance pathways in model span of time. Using the above screening strategy, we
plants.Whileourfunctionalscreenhasbeendesignedto could isolate genes with previously known function in
identify functional genes to abiotic stress tolerance, stress tolerance and novel sequences with unknown
rather than rely on changes in gene expression; several function in salt stress tolerance from J. curcas. The
reports have correlated changes in gene expression to semi-quantitative RT-PCR expression analysis of
possiblegenefunctionsmediatingstresstolerance[8].To selected genes revealed differential expression in leaves
correlate gene function and gene-expression status for and roots in response to salt stress. The isolated genes
eachofthegenes,semi-quantitativeRT-PCRanalysiswas could be over-expressed using plant expression system
conducted. We assayed for changes in transcript abun- to generate and evaluate transgenic plants for stress tol-
danceforeachofthegenesthathavebeennormalizedto erance as well as be used as markers for breeding salt
actin,inbothleafandroottissue,thatwereeithercontrol stress tolerance in plants. With minor modification, the
treatedor saltstressed, beforesamplingatvarioustime- functional screening methodology reported in the pre-
intervals (see Methods). Changes in expression for each sent study may be extended to isolate plant genes that
transcript normalized to actin have been plotted in Fig- confer tolerance to a diverse array of possible abiotic
ure6andFigure7. stresses. The stress conditions that can be screened with
Analysis of the gene-expression data suggests dynamic this assay include a) pH stress (due to acidic or basic
changes in the transcript abundance of these genes, with conditions) b) oxidative stresses c) unfavorable tempera-
changes in transcript level being apparent from 2 h ture d) heavy metal and e) DNA damage/radiation (by
time-point, indicating an early regulation of the genes in exposure to UV, chemicals that induce DNA breakage).
response to salt stress. Gene expression of the normal-
ized transcripts suggests both up-regulation as well as Methods
repression of the transcripts. We noted increased Plant material
expression of profilin in root tissue between 2 h to 8 h Seeds of J. curcas (S003) were collected from Reliance
time-points of exposure to 150 mM salt stress; whereas Life Sciences’ agricultural farm at Kakinada, South of
profilin was repressed in leaves (Figure 7 vs. Figure 6) Andhra Pradesh, India. Seed coats were removed from
consistent with previously reported function during salt the seeds, surface sterilized with 70% ethanol followed
stress [33]. In contrast, we observed an initial repression by 5% hypochloride solution prior to being placed onto
in the expression of annexin that was subsequently MS basal salts [34] distributed into 150-mm culture
induced in the root-tissue, while the expression was tubes. MS basal salts supplemented with 3% sucrose and
down-regulated upon salt-stress in the leaves (Figure 6 solidified with 0.8% agar (Hi-Media, India) were used to
vs. Figure 7). Transcript abundance in the levels of Apx- grow seeds. The cultures were maintained at 23-25°C
1 and metallothioneins were down-regulated in roots, at 50-60% RH, under long day conditions as described
while a more dynamic expression pattern was observed previously [35].
in the leaf tissues, implicating a role in stress adaptive
responses [31]. Among the transcripts we profiled in Stress treatment and sample collection
this study, only LEA-5 was up-regulated in leaves at the Young 3-4 week-old in vitro germinated J. curcas seed-
8 h time interval, after initial down-regulation at the 2 h lings were removed from the media and randomly sepa-
time-point; while in roots LEA-5 expression was unaf- rated into groups of 15-20 plantlets. Roots (both main
fected by salt stress. Such an induction of LEA-5 in leaf- and lateral roots) of these plants were either placed in
tissue demonstrated either a longer range signal or the sterile water or into 150 mM NaCl solution for 2 h. The
terminal effects of long exposure to ions by the roots. treated Jatropha root tissue from plants that were
Further experiments are required to de-lineate these untreated or treated were dissected and frozen in liquid
effects. The gene-expression data, indirectly suggests nitrogen, prior to being stored at -80°C.
Eswaranetal.BMCBiotechnology2010,10:23 Page10of15
http://www.biomedcentral.com/1472-6750/10/23
Figure6Semi-quantitativeRT-PCRanalysisofexpressionoffivedifferentgenesinleaftissueaftertreatingJ.curcasseedlingswith
150mMNaCl.Apx-1,cytosolicascorbateperoxidase-1;Mth,metallothionein;LEA-5,lateembryogenesisabundantprotein-5.BarsrepresentS.E.
ofmean(nvalueis3).
Total RNA isolation columns (BD CloneTech, USA). The yield and quality of
J. curcas root samples control treated or challenged with the amplicons were monitored on agarose gel analysis.
150 mM NaCl were used to generate pools of cDNA. A profile of amplification patterns obtained after 22 and
Total RNA for cDNA synthesis was extracted from 25 cycles of PCR amplifications as described in the
J. curcas root samples using Qiagen RNA Miniprep Kit Super SMART cDNA synthesis Kit [18,36] is shown in
(Qiagen, Germany). Briefly, the root tissue samples were Figure 8. A schematic diagram elaborating the construc-
homogenized to a fine powder in liquid nitrogen and tion of Jatropha root cDNA libraries is outlined (see
total RNA was extracted as described in the Plant mini additional file 2:-process flow).
RNA prep kit (Qiagen, Germany). Total RNA was esti-
mated spectrophotometrically using Nanodrop. Screening for saline tolerant yeast strain and isolation of
shs mutants
Construction of cDNA and expression libraries To identify and define the condition that lead to salt
To prepare poly (A+) mRNA pools suitable for cDNA stress to wild-type yeast BY4741 (See additional file 3:
synthesis, 10 μg total RNA was treated with RNAase genotype details) (yeast strain obtained from EURO-
free DNAaseI (Sigma-Aldrich, St Louis, USA) for 15-20 SCARF, Germany), we measured relative growth of yeast
min at 37°C, and mRNA fraction was enriched using (YPD media 2% Peptone, 1% Yeast extract, 2% Dextrose,
oligo-d(T) beads (Oligotex, Qiagen, Germany). First solidified with 1.5% Agar, Hi-Media, Mumbai, India)
strand cDNA pools were synthesized from normalized under a range of salinity stress, from 0.0 mM NaCl to
amounts of RNA either derived from untreated root tis- 2.0 M NaCl. Salt-hyper-sensitive mutants were isolated
sue or from tissue challenged with salt stress, using through UV induced random mutagenesis and selected
PowerScript reverse transcriptase (Takara, CloneTech) on salt containing plates by replica-plating.
as described in the Super SMART cDNA synthesis Kit
[18]. Double stranded DNA was generated through PCR Yeast functional screening
amplification using conditions described in Table 2 [18]. ToexpressthecDNAlibrariesinyeast,thecDNAlibrary
The cDNA pools were size separated using NucleoSpin amplicons were cloned into a yeast expression vector,