Gene of Pigeonpea Imparts Abiotic Stress Tolerance and Regulates Plant Growth

15 Pages·2017·3.65 MB·English

by  Venkateswara R. Khareedu

ORIGINALRESEARCH
published:10February2017
doi:10.3389/fpls.2017.00165
A Cyclin Dependent Kinase
Regulatory Subunit (CKS) Gene of
Pigeonpea Imparts Abiotic Stress
Tolerance and Regulates Plant
Growth and Development in
Arabidopsis
Editedby: SrinathTamirisa,DashavanthaR.VudemandVenkateswaraR.Khareedu*
AvinashMishra,
CSIR-CentralSalt&MarineChemicals CentreforPlantMolecularBiology,OsmaniaUniversity,Hyderabad,India
ResearchInstitute,India
Reviewedby: Frequent climatic changes in conjunction with other extreme environmental factors are
JulianMarioPeña-Castro,
knowntoaffectgrowth,developmentandproductivityofdiversecropplants.Pigeonpea,
UniversidaddelPapaloapan,Mexico
ChanYulYoo, a major grain legume of the semiarid tropics, endowed with an excellent deep-root
UniversityofCalifornia,Riverside,USA system,isknownasoneoftheimportantdroughttolerantcropplants.Cyclindependent
ChangxunFang,
kinases(CDKs)arecorecellcycleregulatorsandplayimportantroleindifferentaspects
FujianAgricultureandForestry
University,China ofplantgrowthanddevelopment.Thecyclin-dependentkinaseregulatorysubunitgene
GregoryFranklin,
(CKS) was isolated from the cDNA library of pigeonpea plants subjected to drought
PolishAcademyofSciences,Poland
stress.PigeonpeaCKS(CcCKS)geneexpressionwasdetectedinboththerootandleaf
*Correspondence:
VenkateswaraR.Khareedu tissuesofpigeonpeaandwasupregulatedbypolyethyleneglycol(PEG),mannitol,NaCl
[email protected] andabscisicacid(ABA)treatments.TheoverexpressionofCcCKSgeneinArabidopsis
significantlyenhancedtoleranceoftransgenicstodroughtandsaltstressesasevidenced
Specialtysection:
Thisarticlewassubmittedto by different physiological parameters. Under stress conditions, transgenics showed
PlantAbioticStress, higher biomass, decreased rate of water loss, decreased MDA levels, higher free
asectionofthejournal
proline contents, and glutathione levels. Moreover, under stress conditions transgenics
FrontiersinPlantScience
exhibited lower stomatal conductance, lower transpiration, and higher photosynthetic
Received:09December2016
Accepted:26January2017 rates.However,undernormalconditions,CcCKS-transgenicsdisplayeddecreasedplant
Published:10February2017
growth rate, increased cell size and decreased stomatal number compared to those
Citation:
of wild-type plants. Real-time polymerase chain reaction revealed that CcCKS could
TamirisaS,VudemDRand
KhareeduVR(2017)ACyclin regulatetheexpressionofbothABA-dependentandABA-independentgenesassociated
DependentKinaseRegulatorySubunit with abiotic stress tolerance as well as plant growth and development. As such, the
(CKS)GeneofPigeonpeaImparts
CcCKS seems promising and might serve as a potential candidate gene for enhancing
AbioticStressToleranceand
RegulatesPlantGrowthand theabioticstresstoleranceofcropplants.
DevelopmentinArabidopsis.
Front.PlantSci.8:165. Keywords:abioticstresstolerance,abscisicacid,Cajanuscajan,cyclin-dependentkinaseregulatorysubunit,
doi:10.3389/fpls.2017.00165 glutathione,growth,development
FrontiersinPlantScience|www.frontiersin.org 1 February2017|Volume8|Article165
Tamirisaetal. CcCKSRegulatesAbioticStressandDevelopment
INTRODUCTION (Zhou et al., 2013). However, their precise roles in abiotic
stressresponsesarelargelyunknown(KitsiosandDoonan,2011;
Plants often encounter extreme temperatures, drought, and Komaki and Sugimoto, 2012). Therefore, further research will
high salt concentrations in soil, which are the major abiotic helpintheelucidationofpossiblemechanismsofCDKactivity
stresses that negatively affect plant growth and development underdifferenttypesofabioticstresses.
(Krasensky and Jonak, 2012). A prerequisite for plants to In this study, we isolated the stress-responsive Cajanus
acclimatizeunderextremeconditionsistheirabilitytoperceive cajancyclin-dependentkinaseregulatorysubunitgene(CcCKS)
stress signals and transduce them through a chain of signaling from pigeonpea. The overexpression of CcCKS in Arabidopsis
molecules,whichultimatelyinteractwiththeregulatoryelements conferreddroughtandsalinitystresstoleranceaswellasmarked
of different stress-inducible genes and bring about changes in changes in the growth and development of transgenic plants.
cellular gene expression (Kasuga et al., 1999; Tong et al., 2007; ThisreportclearlydemonstratesthattheCcCKSinArabidopsis
Tamirisaetal.,2014).Anunderstandingofthemechanismsthat affords abiotic stress tolerance and regulates plant growth and
regulate the expression of stress-responsive genes is essential development.
for enhancing plant productivity under adverse environmental
conditions(Priyankaetal.,2010a,b).
MATERIALS AND METHODS
The development of plant organs directly depends on the
frequency of cell division, parameters of the cell cycle, and Plant Materials and Stress Treatments
number and size of the cells. These developmental processes
The pigeonpea seeds (variety ICP 8744) were disinfected with
are controlled by the molecular machinery that regulates cell
0.1% (w/v) mercuric chloride and germinated in Petri plates.
cycle progression in coordination with nutritional, hormonal,
Four-week-old plants were subjected to polyethylene glycol
developmental and environmental signals (Inze and Veylder,
(PEG) 6000 (20% w/v), mannitol (400mM), NaCl (1.0M), and
2006; Tank and Thaker, 2011). A central role in the regulation
ABA(100µM)for6and12h.ThesterilizedseedsofArabidopsis
of the cell cycle is played by a group of proteins known as ◦
(Col-0), after stratification for 3 days at 4 C, were grown on
cyclin-dependent kinases (CDKs), a family of protein kinases MurashigeandSkoog(1962)at20±1◦Cwitha16hphotoperiod
that were indentified for their role in regulating the cell cycle. under fluorescent light (120–150 µmol/m2 sec) in a Conviron
CDKsarecorecellcycleregulatorsthatareinvolvedinvarious
growthchamber(Tamirisaetal.,2014).
processes of plant growth and development (Ma et al., 2015)
andhavebeenimplicatedinadditionalcellularprocessessuchas Subtraction cDNA Library Construction
transcription and translation. A progressive transition through
Total RNA was isolated from 4-week-old 20% PEG-treated
different phases of the cell cycle is controlled by a conserved
(−1.01±0.02Mpa)andunstressed(−0.49±0.02Mpa)plants
mechanism based on the sequential formation and activation
bytheguanidiniumthiocyanatemethod(SambrookandRussell,
of complexes between CDKs and their activating cyclin (CYC)
2001).Poly(A+)RNAwaspurifiedfromthetotalRNAthrough
subunits (De Veylder et al., 2003). CDK subunit proteins
oligo (dT) cellulose chromatography using an mRNA isolation
(CKS) might act as both activators and inhibitors of CDK
kit(AmershamPharmaciaBiotech,AsiaPacificLtd,QuarryBay,
activity (De Veylder et al., 2001a). CKS regulate CDK activity
Hong Kong). The PCR select cDNA subtractive hybridization
by functioning as docking factors for the CDK/CYC substrate
method was used to construct the cDNA library (Clontech,
complexes(Borucetal.,2010a).TheSchizosaccharomycespombe
Mountain View, CA, USA) with 2 µg of mRNA isolated from
SUC1genewasidentifiedinfusionyeastbyitsabilitytorescue
controlandPEG-treatedplants(Tamirisaetal.,2014).
certaintemperature-sensitivecdc2mutants(Haylesetal.,1986),
and its homolog, the Saccharomyces cerevisiae CKS1 gene was
Isolation of Full-Length Cajanus cajan
found to be a suppressor of CDC28 mutants (Hadwiger et al.,
Cyclin-Dependent Kinase Regulatory
1989). TheSUC1/CKS1 homolog was isolated from Arabidopsis
using yeast two-hybrid screening and designated as AtCKS1 Subunit Gene (CcCKS)
(De Veylder et al., 1997). The overexpression of AtCKS1 in Total RNA was isolated from the PEG (20%) treated 4-week-
Arabidopsisdecreasedtheleafsizeandreducedtherootgrowth old plants as described above. RT-PCR for first-strand cDNA
ratewithoutanyeffectonendoreduplication(DeVeylderetal., synthesis was carried out using a reaction mixture containing
2001a). Tris-HCl(10mM),KCl(50mM),MgCl (1.5mM),dNTPs(200
2
Under biotic and abiotic stress conditions, plant growth is µMeach),MMLVreversetranscriptase(2units),totalRNA(1.0
adversely affected as a result of cell cycle inhibition due to a µg), and Oligo (dT) primer. The resultant cDNA product was
prolonged S phase and delayed entry into mitosis (Kitsios and usedasatemplateinaPCRreactionusinggene-specificprimers
Doonan, 2011). Though the molecular interactions linking the to amplify the CcCKS gene. The full-length coding sequence
cell cycle machinery to the perception of stress signals are not (267 bp) of CcCKS, amplified using gene-specific primers and
fullyunderstood,recentstudieshaveindicatedtheinvolvement using the drought-stressed cDNA library as a template, was
of CDKs in the plant stress responses. An Arabidopsis CYCH;1 ligatedattheSmaI-siteofthepBSSK(+)vectorandtransformed
genewasreportedtobeinvolvedindroughtstressresponseby into E. coli (TOP10) cells. The resultant recombinant clones
regulating the blue light-mediated stomatal opening as well as were selected and confirmed by restriction digestion with XhoI
by controlling the reactive oxygen species (ROS) homeostasis and XbaI enzymes and later sequenced using an automated
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Tamirisaetal. CcCKSRegulatesAbioticStressandDevelopment
DNAsequencer.Homologysearchofnucleotideandaminoacid Construction of the CcCKS:GFP Fusion
sequences was carried out using BLAST (NCBI). A maximum Gene Construct and Generation of
likelihoodphylogenetictreewasconstructedbasedontheamino Transgenic Arabidopsis Plants
acid sequences of CcCKS protein, and other CKS proteins
The CcCKS coding sequence without a termination codon
from different plant species, using Clustal Omega and MEGA6 was fused with the 5′ region of green fluorescent protein
software.Thebootstrapparameterwassetat100.
(GFP) and the fusion gene was driven by the CaMV35S
promoter. The expression cassettes of pCaMV35S–
Plant Expression Cassettes Construction
CcCKS:GFP/pCaMV35S:GFP were subcloned into pBI101
and Transformation of A. thaliana
containing the nptII expression unit and mobilized into the
Plasmid DNA containing the CcCKS gene was digested with Agrobacterium (EHA105 strain) through triparental mating.
XhoI-XbaI enzymesandwassubcloned intothepRT100vector The transformed seedlings were selected on MS medium
at the 3′ end of the CaMV35S promoter. Furthermore, the supplementedwithkanamycin(50mgL−1).Calluswasinduced
gene cassette was digested with HindIII and cloned into the fromtherootsofpCaMV35S:GFPandpCaMV35S–CcCKS:GFP
pBI101 vector. The pBI101 vector containing CcCKS and nptII transgenics on 2, 4-dichlorophenoxyacetic acid (2 mgL-1)
expression units and the pBI101 vector with nptII (empty containingmedium.Individualcalluscellswerevisualizedunder
vector) were mobilized independently into the EHA105 strain confocal microscopy (Leica SP5 Microsystems, Excitation: 488,
ofAgrobacteriumthroughtriparentalmating(Wiseetal.,2006). andEmission:520).
The Agrobacterium-mediated floral dip method was carried
out to transform A. thaliana (Clough and Bent, 1998). Seeds Functional Analysis of Transgenics for
from the transformed plants were germinated on MS medium
Abiotic Stress Tolerance
supplementedwithkanamycin(50mgL−1)toselecttheputative
The seeds of WT, VT and homozygous transgenic (T )
transformants. 3
ArabidopsisplantsofCS1,CS2andCS3weresurface-sterilized,
Molecular Analysis of CcCKS Transgenic placed on MS salt medium, and allowed to grow as described
above. Drought and salt tolerance of 2-week-old transgenic
Arabidopsis Plants
seedlings was tested by growing them on MS medium
GenomicDNAisolatedfromkanamycintolerantplantswasused
supplemented with mannitol (150mM) or NaCl (125mM) for
to carry out PCR analysis. DNA isolated from untransformed
7 days. Later, the seedlings were transferred onto the MS salt
plants (WT) was used as a negative control and plasmid
medium and allowed to recover for 10 days under normal
(pBI12135SnptII-p35SCcCKS) DNA was used as a positive
conditions. After 10 days of recovery, the survival rate, root
control.ForPCR,plasmidDNA(10ng)andgenomicDNA(50
length and biomass of the seedlings were recorded. Another
ng) were used as templates in separate reactions. The reactions
set of stressed seedlings were transferred to the soil, grown
were carried out using CcCKS gene-specific primers, and the
for maturity and photographed. For drought tolerance assays,
amplifiedPCRproductswereanalyzedona1.0%agarosegel.
3-week-old plants growing in pots were withheld from water
for 2 weeks, and then rewatered for 1 week (Sekhar et al.,
Southern Blot Analysis of Arabidopsis
2010).
Transgenic Plants
GenomicDNA(15µg)wasisolatedfromthreeCcCKStransgenic Relative Water Content Measurements
plantsbytheCTABmethod(Saghai-Maroofetal.,1984),andwas
The relative water contents (RWC) of 4-week-old transgenic
digested independently with the HindIII enzyme. The digested
plants of CS1, CS2 and CS3 along with WT and VT plants
DNA was resolved on 0.8% agarose gel and transferred to a
was measured. Fresh weight loss was calculated relative to the
positively charged nylon membrane as per the manufacturer’s
initial plant weight. To measure the fresh weight, plants were
instructions (Amersham Pharmacia Biotech Asia Pacific Ltd, weighed immediately and left in the growth chamber (20 ±
Quarry Bay, Hong Kong). A Southern blot analysis was ◦
1 C)untiltherewasnofurtherweightloss(desiccatedweight).
performedaccordingtoSambrookandRussell(2001).ThenptII ◦
Finally, the plants were dried for 24 h at 70 C and the dry
codingregionwaslabeledwiththeAlkPhosDirectLabelingand
weightswererecorded.Therelativewatercontentofthesamples
Detection System according to the manufacturer’s instructions was measured using the formula RWC (%) = (desiccation
(Amersham Pharmacia Biotech Asia Pacific Ltd, Quarry Bay, weight—dry weight)/(fresh weight–dry weight) × 100 (Mao
HongKong)andusedasaprobe.
etal.,2010).
RT-PCR Analysis CcCKS Transgenic
Osmotic Potential Analysis
Arabidopsis Plants
Two-week-oldplantsofCS1,CS2,andCS3alongwithWTand
Total RNA was extracted from transgenic (CS1, CS2 and CS3), VTplantswereselectedandfrozenwithliquidnitrogenfor30s,
wild-type (WT) and vector control (VT) plants using the and preserved at −80◦C. Sap was collected by squeezing the
TRIZOL(Invitrogen,Carlsbad,CA,USA).RT-PCRwascarried leaves using a micro pestle, and the osmotic potential of the
out as described earlier, and the amplified PCR products were sap was determined using a vapor pressure osmometer (Vapro,
analyzedon1.0%agarosegel. Model5520;WescorInc.,Logan,UT,USA;Maoetal.,2010).
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Tamirisaetal. CcCKSRegulatesAbioticStressandDevelopment
Cell Membrane Stability Analysis Effect of ABA on Seed Germination and
Ten-day-old seedlings grown on 1X MS medium were placed Seedling Growth
on filter paper saturated with NaCl (150mM) solution. As SeedsoftransgenicandWTplantswereplacedonMSmedium
soon as symptoms of stress appeared (appx. after 6h) in WT containingdifferentconcentrationsofABA(0–2.0µM)torecord
and VT plants, the seedlings were removed, rinsed thoroughly the germination frequency. Further, germinated seedlings were
and immersed in 20ml of double distilled water at room allowedtogrowfor20daystoassesstheirsensitivitytoABA.
temperature(20±1◦C).Theinitialconductivityofsampleswas
recorded after 2h using a conductivity meter (Model Sension Measurement of Stomatal Aperture Sizes
5; HACH Company, Loveland, CO, USA). Later, samples were Rosette leaves of 3-week-old plants were detached and floated
boiled for 30min and, cooled to room temperature, and the (abaxial side down) on solution containing 10mM MES-KOH
finalconductivitywasmeasured.CMSwascalculatedusingthe (pH 6.15), 30mM KCl and 1mM CaCl and were incubated
2
formula CMS (%) = 1—initial electrical conductivity/electrical underlightfor2h.Later,theyweretreatedwithABA-containing
conductivityafterboiling×100(Maoetal.,2010). solution for 2h. In each treatment, 50 stomatal aperture sizes
weremeasuredunderconfocalmicroscopy.
Measurement of the Leaf Chlorophyll
Content Root Growth Rate
Leaf disks of CS1, CS2, and CS3 transgenic lines along with For root growth rate analysis, the seeds were germinated on
WT and VT plants were floated independently on water MS medium and placed vertically in the growth chamber. The
(control),150mMmannitoland125mMNaClsolutionsatroom position of the root tip was marked everyday on the backside
temperature (20 ± 1◦C) for 72h. Leaf disks were then used of the Petri plates to determine the daily growth. After 20
to measure the chlorophyll content spectrophotometrically as days, the plates were scanned using a WinRHIZO standard
describedbyTamirisaetal.(2014). systemsdesktopopticalscanner.Thedailylengthincreaseover
the entire growing period was determined by measuring the
Measurement of the Proline Content
distancebetweensuccessivemarksalongtherootaxisusingthe
Fifteen-day-oldCcCKStransgeniclinesalongwithWTandVT WinRHIZOsoftware.Bydividingthedailygrowthbytheprecise
seedlingswereexposedto150mMmannitoland125mMNaCl time interval between the corresponding marks, the average
for 3 days. Plant tissues were homogenized in 3% aqueous growthratewascalculated(DeVeylderetal.,2001a).
sulfosalicylicacidandthehomogenateswerecentrifugedat1000
rpmfor5min.equalvolumesofglacialaceticacidandninhydrin Flow Cytometric Analysis
wereaddedtothesupernatantcollected.Theprolinecontentwas The DNA content was estimated by flow cytometric analysis.
quantifiedasdescribedbyBatesetal.(1973). Plantmaterials(50mg)oftrangenicandWTplantsweretaken
and chopped using a razor blade into 1ml of ice-cold Otto I
Measurement of the Malonaldehyde (MDA)
buffer in a Petri plates. The solution with the chopped plant
Content tissue (suspension) was filtered through 40 µm nylon mesh;
Tissue (50 mg) was collected from stressed and unstressed later, nuclei were pelleted by centrifugation (150 g/5min). The
transgenics, WT and VT seedlings and homogenized with 2ml nuclei were resuspended by gentle shaking and 100 µl of fresh
of 0.1% (w/v) cold trichlorocactic acid (TCA) on ice. The OttoIbufferwasadded.Thesampleswereincubatedfor60min.
homogenateswerecentrifugedat14,000×gfor10minat4◦C, Afterincubation,OttoIIbuffer(1ml)whichincludespropidium
after which 250 µl of supernatant was mixed with 1.5ml of iodide(50µg/ml)andRNase(50µg/ml)wasadded.Thesamples
TCA/thiobarbituric acid (0.25% thiobarbituric acid containing wereleftatroomtemperatureandanalyzedafter20minusinga
10%TCA)reagent.TheMDAcontentwasestimatedaccording GuavaEasyCyteflowcytometer(Millipore,France;Dolezeland
toShinetal.(2012). Bartos,2005).
Detection of Superoxide Radicals in WT Pollen Staining
and Transgenic Plants Pollen staining was performed using the pollen collected from
Thenitrobluetetrazolium(NBT)stainingmethodwasusedfor newlydehiscentflowersasdescribedbyAlexander(1969).
super oxide detection. Three-week-old transgenic, WT and VT
Measurement of the Glutathione (GSH)
plants were used for NBT staining. Plants were germinated on
MS basal medium and later transferred to medium containing Content
150mMmannitol(droughtstress)and125mMNaCl(saltstress) The GSH levels were measured in the intact roots and guard
for3days,alongwithunstressedseedlings.Thetreatedseedlings cells of Arabidopsis after conjugation with monochlorobimane
werevacuum-infiltratedwith0.1mg/mlNBTin25mMHEPES (MCB), to produce a fluorescent glutathione S-bimane (GSB)
buffer, pH 7.6. The samples were incubated in the dark for 2h adduct. Seedlings that were treated with 150mM mannitol for
inagrowthchamberandlaterstainedsamplesweretransferred 2 days were carefully removed from the agarose surface, and
to 80% ethanol and incubated at 70◦C for 10min to remove therootswereplacedinadropof100µMmonochlorobimane.
chlorophyll. For each treatment, 20 seedlings of each line were After labeling for 15min, the roots were washed briefly in
used. distilled water and mounted on a microscope slide with
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Tamirisaetal. CcCKSRegulatesAbioticStressandDevelopment
a cover slip. To measure the GSH levels in the guard qRT-PCR are listed in SupplementaryTable1 (Tamirisa et al.,
cells, excised leaves from treated plants were incubated in 2014).
a staining solution containing 100 µM of MCB for 2h at
room temperature. Photographs were taken using a fluorescent Statistical Analysis
microscope (Olympus BX41fluorescence microscope). The All the experiments were done in triplicate using 20 seedlings
captured images were used to measure the number of pixels/ in each treatment. Fresh tissue was collected for all the
fluorescenceintensityusingImageJsoftware. experiments. Mean, standard error and t-test were computed
with the help of pre-loaded software in Excel, for statistical
Histological Analysis
calculations.
The cell density, stomatal density and stomatal index were
determined as described by De Veylder et al. (2001a,b). Leaves
RESULTS
of the same age and same relative position were sampled for
analysis, and the leaf surface imprint method was used as Characterization of the CcCKS Gene
described by Yu et al. (2008). Three leaves were sampled from
A cDNA clone of 267 bp sequence (KU522130), encoding a
each plant, and 10 of each WT, VT and transgenic plants were
polypeptide of 88 amino acids, was designated as the cyclin-
sampled.
dependent kinase regulatory subunit gene (CKS). Phylogenetic
tree analysis revealed that the CcCKS protein was clustered
Scanning Electron Microscopy
in the same monophyletic group as putative CKS proteins
Cotyledons from 3-week-old seedlings were processed and
from Glycine max, Medicago truncatula and Arabidopsis
analyzed using scanning electron microscopy, as described
lyrata (SupplementaryFigureS1). Further, the CcCKS protein
De Veylder et al. (2002). Seedlings were fixed overnight
revealed high similarity with CDKs regulatory subunits of G.
in 4% paraformaldehyde and 1% glutaraldehyde in 0.1M
max (99%), M. truncatula (97%), A. lyrata (95%), Nicotiana
phosphate buffer (pH 7.2), followed by post-fixation in 2%
tomentosiformis(95%),Zeamays(90%),A.thaliana(90%),and
osmiumtetroxide, and a graded ethanol series. These seedlings
Brassica rapa (87%). To examine the subcellular localization
weremountedonstubs,sputter-coatedwithgoldandexamined
of the CcCKS protein, pCaMV35S-CcCKS:GFP fusion and
usingascanningelectronmicroscope(Zeiss,Germany).
pCaMV35S:GFPgene(control)constructswereintroducedinto
Measurement of Stomatal Conductance, Arabidopsis and the individual callus cells were visualized
under laser-scanning confocal microscopy. Cells expressing the
Transpiration, Photosynthetic Rates, and
CcCKS:GFPfusionproteinshowedthefluorescencebothinthe
Water use Efficiency
nucleusandthecytoplasm(SupplementaryFigureS2).
The stomatal conductance, transpiration and photosynthetic
ratesweremeasuredusingaportablephotosynthesissystem(Li- Expression Profile of CcCKS under Abiotic
Cor LI-6400XT). Four-week-old plants growing on soil were
Stress Conditions in Pigeonpea
treated separately with 150mM mannitol and 125 mM NaCl
To analyse the stress-inducible nature of the CcCKS gene, real-
for 3 days. All of the photosynthetic measurements were taken
at a constant air flow rate of 500 mmol s−1. Furthermore, the timePCRwasdoneusingthetotalRNAisolatedfrompigeonpea
concentration of CO (400 mmol mol−1 using the system’s plants after treatment under different stress conditions. These
2
CO injector), temperature (22◦C), and photosynthetic photon results revealed that increased levels of CcCKS transcripts were
2
flux density (1,200 mmol (photon) m−2 s−1) were maintained. detected in both the roots and leaves of PEG-, mannitol-,
NaCl- , and ABA- treated plants compared to untreated plants
Wateruseefficiencywascalculatedusingthephotosyntheticand
(Figure1).
transpirationmeasurements.Threemeasurementsweretakenon
eachplant,and10plantsfromWT,VTandeachtransgenicline
Development of CcCKS-Transgenic Plants
wereusedformeasurements.
of A. thaliana
Quantitative Real Time PCR (qRT-PCR) Putative transgenic plants, obtained with plasmid constructs
qRT-PCR was performed using RNA isolated from stressed containing CcCKS (Figure2A), were selected on MS medium
and unstressed pigeonpea plants as described earlier. RNA was containing kanamycin (50mg L−1). PCR using CcCKS gene-
isolated from WT and transgenic plants subjected to mannitol specific primers amplified an ∼250 bp fragment from the
(150mM)stressfor2daysaswellasfromunstressedplantsas genomic DNA of transgenic plants, while no such band was
describedearlier.FirststrandcDNAwassynthesizedfromRNA observed in WT (Figure2B). RT-PCR revealed the presence
samplesandusedasatemplateforqRT-PCR.qRT-PCRanalysis of CcCKS transcripts in pCaMV35S-CcCKS lines (Figure2C).
was performed using SYBR green master mix with the Applied A Southern blot analysis of the genomic DNA isolated from
Biosystems7500RealTimePCRsystem(USA).Thegeneswere transgenic lines demonstrated the existence of the transgene
selectedbasedontheliteratureavailableandthespecificprimers in the genome of Arabidopsis, when probed with nptII
were designed. Each reaction was performed thrice, and the (Figure2D).HomozygoustransgeniclinesofCS1,CS2andCS3,
relative expression ratio was calculated using 2−11ct method containingasingleT-DNAinsertion,werechosenforsubsequent
withactinasthereferencegene.Theprimersthatwereusedfor experiments.
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Tamirisaetal. CcCKSRegulatesAbioticStressandDevelopment
FIGURE1|AnalysisofCajanuscajancyclindependentkinaseregulatorysubunitgene(CcCKS)expressioninpigeonpeaunderdifferenttreatment
conditionsbyqRT-PCR.ComparisonoftherelativetranscriptlevelsofCcCKSgeneunder(PEG)6000(20%w/v),Mannitol(400mM),NaCl(1.0M),andABA(100
µM)stressfor6h(A)and12h(B).Actinhasbeenusedasreferencegene.Theverticalcolumnindicatestherelativetranscriptlevel.Barrepresentsmeananderror
barsrepresentsSEfromthreeindependentexperiments.AsterisksindicatesignificantdifferencesincomparisonwiththeWTatP<0.05.WSrepresentswithout
stress.
FIGURE2|T-DNAregionofpBI101containingCcCKSexpressioncassetteandmolecularconfirmationofCcCKStransgenicArabidopsisplants.
(A)RestrictionmapofT-DNAregionofpBI101containingCcCKSexpressionunitdrivenbyCaMV35SpromoterandnptIIselectionmarker.(B)PCRanalysisforthe
presenceofCcCKSgeneinthegenomicDNAoftransgenicArabidopsisplants.LaneM:1.0kbDNAsizestandardmarker,LaneP,Positivecontrol;LaneWT,wild
type;Lanes1–3,PCRamplificationofCcCKSfromtransgenicplants.(C)RTPCRanalysisshowingtheexpressionofkanamycinandCcCKSgenesintransgenic
Arabidopsis.LaneWT,wild-type;LaneVT,vectorcontrol;Lanes1,2,and3:differenttransgeniclines.(D)SouthernblotanalysisoftransgenicArabidopsisplants
carryingCcCKSgene.LaneWT,wildtypeplant;Lanes1,2,and3:transgenicplantsofthreedifferentlines(CS1,CS2,andCS3).
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Tamirisaetal. CcCKSRegulatesAbioticStressandDevelopment
Functional Analysis of CcCKS-Transgenic to reach the reproductive phase under drought and salt stress
Lines for Abiotic Stress Tolerance conditions (Figure4). The transgenic plants subjected to low
water conditions also showed greater tolerance compared to
No variation was observed in the seed germination of
theWTandVTplants(SupplementaryFigureS3A).Although
transgenic,VTorWTseedsundernormalconditions.However,
waterlosseswereobservedintransgenicandcontrolplants,the
under mannitol (150mM) and NaCl (125mM) stress, higher
finalrelativewatercontentoftransgenicswassignificantlyhigher
germination rates were found in transgenic plants than in WT
than that of WT and VT plants (SupplementaryFigureS3B).
or VT plants (Figure3A). To characterize the stress tolerance
When the osmotic potential of transgenic, VT, and WT plants,
abilityoftransgenicplants,2-week-oldseedlingsweresubjected
grown under well-watered conditions was measured, the CS1,
to drought (150mM mannitol) and salt (125mM NaCl) stress
CS2,andCS3linesshowedhigherosmoticpotentialthaninVT
treatments for 7 days along with VT and WT seedlings.
andWTplants(SupplementaryFigureS3C).
Transgenic lines of CS1, CS2, and CS3 exhibited increased
survivalrate,biomassandrootlengthscomparedtoVTandWT Estimation of the Proline, Malonaldehyde
plants(Figures3A–D).
(MDA), and Glutathione Levels and the Cell
Under normal conditions, no significant differences in
Membrane Stability in CcCKS-Transgenic
chlorophyll contents were observed between the control and
transgenic plants. The transgenic plants showed higher mean Plants
chlorophyll contents under both mannitol and NaCl stresses To understand the physiological basis of stress tolerance of
compared to those of WT and VT plants (Figure3E). Further, CcCKS-transgenic plants, the proline content was estimated
the transgenic plants expressing CcCKS gene could successfully under normal and stress conditions. Under normal conditions,
completetheirreproductivecycle,whilethecontrolplantsfailed transgeniclinesshowedenhancedprolinelevels,whiletherewas
FIGURE3|EvaluationofCcCKS-transgenicsagainstdifferentabioticstresses.Seedgerminationrate(A)ofWT,VT,andtransgenicplantsundernormal,
150mMmannitoland125mMNaClstress.Two-week-oldseedlingsofWT,VT,andtransgenicsweregrownon150mMmannitoland125mMNaClfor7daysand
werelaterallowedtorecoveronMSplates.Dataonsurvivalrate(B),totalbiomass(C)androotlength(D)wererecordedafter10daysofrecovery.Ineachtreatment,
20seedlingsofWT,VT,andtwotransgeniclineswereused.Chlorophyllcontent(E)wasdeterminedfromtheleafdisksofcontrolandtransgenicsafter72hof
incubationinwater,150mMmannitoland125mMNaClsolutionsindependentlyatroomtemperature(20±1◦C).BarrepresentsmeananderrorbarsrepresentsSE
fromthreeindependentexperiments.AsterisksindicatesignificantdifferencesincomparisonwiththeWTatP<0.05.WS,withoutstress;CS1,CS2,andCS3:three
differenttransgeniclines;WT,wildtype;VT,vectortransformedplants.
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Tamirisaetal. CcCKSRegulatesAbioticStressandDevelopment
FIGURE4|EvaluationoftransgenicplantsexpressingCcCKSgeneagainstdifferentabioticstressconditions.Two-week-oldseedlingsofWT,VT,and
CcCKS-transgenicsweresubjectedto150mMmannitol,125mMNaClfor7days.Ineachtreatment20seedlingswereused.Treatedseedlingswereallowedto
recoverfor7daysat20±1◦Ctemperature.Later,seedlingsweretransferredtosoilandallowedtogrowfor3weeksundernormalconditions,andwere
photographed.WT,wildtype;VT,vectortransformed;CS1,CS2,andCS3:threedifferenttransgeniclines.
nosignificantdifferencebetweenWTandVTplants.When15- unstressedconditions,transgenicandcontrolseedlingsrevealed
day-old transgenics were subjected to mannitol (150mM) and alowintensityofstaining.However,understressconditions,the
NaCl (125mM) stresses, they could accumulate higher proline transgenicsexhibitedalowerstainingintensitythanthatofWT
contents(Figure5A),whereas,WTandVTplantsshowedlower andVTseedlings,asvisualizedbythepurpleformazandeposit
levelsofprolineundersimilarstressconditions(Figure5A). (Figure6).
TheMDAcontentsintransgenicplantswassignificantlylower
ABA Sensitivity of CcCKS-Transgenic
than those of WT and VT plants when subjected to mannitol
andNaClstressconditions(Figure5B).However,therewasno Plants and Their Stomatal Aperture Sizes
significantdifferencebetweenWTandVTplantsundersimilar The seed germination rate of transgenic plants was
stressconditions(Figure5B). severely inhibited by ABA compared with the WT
To analyze the cell membrane stability, transgenic, WT, and plants (SupplementaryFigureS4A). Transgenics when
VTplantsweretreatedwithNaCl(150mM).Transgenicplants treated with ABA, showed a greater reduction in the
showedsignificantlyhighermembranestabilitythanthatofWT stomatal aperture size compared to that of WT plants
and VT plants. However, there was no difference between WT (SupplementaryFigureS4B).Furthermore,transgenicseedlings
andVTplantsundersimilarstressconditions(Figure5C). showedgreaterhypersensitivitytoABAthanthatofWTplants
Monochlorobimane (MCB) staining was performed to (SupplementaryFigureS4C).
measure the GSH contents in the roots and guard cells of
Stomatal Conductance, Transpiration,
Arabidopsis.Understressconditions,thefluorescenceintensityof
GSHintheguardcellsoftransgenicplantswasmuchhigherthan Photosynthetic Rates, and Water Use
that of WT plants (Figures5D,E). Similarly, GSH fluorescence Efficiency under Stress Treatments
intensityintherootsoftransgenicplantswashigherthanthatof
Under normal conditions, the transgenic lines showed lower
WTplants(Figures5F,G).
stomatal conductance, transpiration and photosynthetic
rates compared to WT plants. However, transgenics
Detection of Reactive Oxygen Species by
subjected to mannitol (150mM) and NaCl (125mM)
NBT Staining stresses, exhibited lower stomatal conductance, lower
The superoxide concentration in the 2-week-old seedlings, transpiration, higher photosynthetic rates and water
treated with mannitol (150mM) and NaCl (125mM), was use efficiency. By contrast, WT and VT plants showed
examined using nitroblue tetrazolium (NBT) staining. Under higher stomatal conductance, greater transpiration and
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Tamirisaetal. CcCKSRegulatesAbioticStressandDevelopment
FIGURE5|Measurementofproline,MDA,osmoticpotential,cellmembranestabilityandrelativewatercontentsoftransgenicplants.Two-week-old
seedlingsofWT,VT,andtransgenicsweregrownon150mMmannitolfor3days,forestimationofproline(A),MDA(B).Cellmembranestability(C)oftransgenics,
WTandVTseedlingsweretreatedwith150mMNaCl.Two-week-oldA.thalianaseedlings,grownonMSmedium,weresubjectedtomannitol(150mM)for2days.
GSHcontentinguardcells(D,E)androotmeristem(F,G)ofWTandtransgenicplant.TrepresentsCcCKS-transgenicandWTrepresentswild-type.Barrepresents
mean,anderrorbarsrepresentsSEfromthreeindependentexperiments.Foreachtreatment,20seedlingswereused.Asterisksindicatesignificantdifferencesin
comparisonwiththeWTatP<0.05.WT,wildtype;VT,vectortransformed;CS1,CS2,andCS3:threedifferenttransgeniclines.(D)Scalebar50µm;(F).Scalebar
100µm.
lower photosynthetic rates under similar stress conditions Developmental Changes in CcCKS
(Figures7A–D). Transgenics in the Root Growth Rate, Leaf
Size, Pavement Cell Number, Stomatal
Pollen Viability Number, Stomatal Index, and Stomatal
Fertile pollen grains were observed in both transgenic Morphology
and WT plants without any differences in the
Three weeks after seed germination, WT, VT, and CcCKS
pollen fertility as evidenced by Alexander staining
transgenic plants exhibited distinct observable differences in
(SupplementaryFigureS5A).
theirmorphologicalfeatures.Transgenicplantsshowedsmaller
rosettes and leaves compared to WT plants (Figure8A). There
Flow Cytometric Analysis were no observable differences in the root length of transgenic
To determine whether the cell cycle was affected in the and WT seedlings during the initial 3 days of root growth.
CcCKS overexpressing plants, flow cytometric analysis was Later, CcCKS transgenic lines showed an approximately steady
performed on the nuclei isolated from the roots of WT and growth rate, while WT plants showed accelerated root growth
transgenic plants. Wild-type roots of Arabidopsis showed the during the 15 day growth period analyzed (Figure8B). The
typical patterned peaks ranging from 2C to 8C, and the C pavementcelldensityontheadaxialsurfaceinsimilarleafareas
values of transgenic lines were similar to those of WT plants from CS1, CS2, and CS3 transgenics was lower than that of
(SupplementaryFigureS5B). WT and VT plants (Figure8C). The size of cells leaving the
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Tamirisaetal. CcCKSRegulatesAbioticStressandDevelopment
FIGURE6|Superoxidedetectionintransgenicplantssubjectedtodifferentabioticstresses.Two-week-oldA.thalianaseedlingsgrownonMSmedium
weresubjectedtomannitol(150mM)andNaCl(125mM)for72h.Foreachtreatment,20seedlingswereused.NBTstainingwasdoneforsuperoxidedetection.WT:
wildtype;VT:vectorcontrol;CS1,CS2,andCS3:threedifferenttransgeniclines.
meristemwasgreaterinCcCKStransgenics(85±7.87µm)than DISCUSSION
in WT (47 ± 3.54 µm). The results clearly showed significant
differences between transgenics and WT plants (P < 0.05). A stress-responsive cyclin-dependent kinase regulatory subunit
Moreover, the mature cell length in the transgenic roots was gene(CKS)wasisolatedfromthecDNAlibraryconstructedusing
slightly longer (191 ± 8.96 µm) than in WT cells (175 ± 10.1 the RNA isolated from drought stressed pigeonpea plants. The
µm). CcCKSproteinstructureanalysisindicateditssimilaritytoother
Compared with WT and VT plants, transgenics revealed CKS proteins of G. max, M. truncatula, A. lyrata, Z. mays, N.
a substantially lower stomatal density and stomatal index tomentosiformis,andB.rapa.
(Figures8D,E). The morphology of stomata in WT and HigherlevelsofCcCKSexpressionweredetectedbothinthe
CcCKS-transgenic Arabidopsis plants appeared normal. leavesandrootsunderdrought,ABAandsaltstresstreatments,
However, scanning electron micrographs clearly demonstrated compared to unstressed plants, indicating the stress-responsive
a few (3%) abnormal stomata in the transgenic plants nature of the gene. It was previously reported that the maize
(Figure8F). cyclin-dependent kinase regulatory subunit gene (ZmCKS2)
showed variable expression levels in different parts of the
seedlings,andwasupregulatedbydroughtandMeJAtreatments
Analysis of Expression Levels of Selected
anddownregulatedbycoldorABAtreatments(Baoetal.,2010).
Genes in CcCKS-Transgenic Plants by
ThelocalizationoftheCcCKS:GFPfusionproteininboththe
qRT-PCR nucleusandthecytoplasm(SupplementaryFigureS2)suggests
Under stress conditions, real time PCR analysis revealed that the CcCKS protein can enter the nucleus and it might
substantialincreasesintheexpressionlevelsofcertaingenes,viz., interact with different proteins to regulate the expression of
delta-1-pyrroline 5-carboxylase synthetase (P5C1), bHLH129 various genes involved in the plant development and stress
transcription factor, MYB44 transcription factor, glutathione response.ArabidopsisCKS1andCKS2subunitswerehighlyand
synthetase (gsh2), and glutamate cysteine ligase (gsh1), in homogenously expressed in the nucleus and cytoplasm (Boruc
transgenic plants compared to those of WT plants. However, etal.,2010b).
the genes encoding cyclin H1, cyclin-dependent kinase B1, Under normal conditions, CcCKS transgenic plants were
MYB60 transcription factor and bHLH128 transcription smaller in size with a significant reduction in fresh weight,
factor were significantly downregulated in transgenics in addition to smaller rosettes and leaves. Transgenics also
under similar stress conditions compared to WT plants exhibited fewer epidermal cells per unit leaf area (Figure8).
(Figure9). In transgenics, the cells leaving the root meristem were larger
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