Overexpression of a Cytosolic Abiotic Stress Responsive Universal Stress Protein (SbUSP

21 Pages·2016·3.38 MB·English

fpls-07-00518 April19,2016 Time:16:47 #1
ORIGINALRESEARCH
published:21April2016
doi:10.3389/fpls.2016.00518
Overexpression of a Cytosolic
Abiotic Stress Responsive Universal
Stress Protein (SbUSP) Mitigates Salt
and Osmotic Stress in Transgenic
Tobacco Plants
PushpikaUdawat1,2,RajeshK.Jha1,2,DinkarSinha1,AvinashMishra1,2*and
BhavanathJha1,2*
1MarineBiotechnologyandEcologyDivision,CSIR-CentralSaltandMarineChemicalsResearchInstitute,Bhavnagar,India,
2AcademyofScientificandInnovativeResearch,CouncilofScientificandIndustrialResearch,NewDelhi,India
The universal stress protein (USP) is a ubiquitous protein and plays an indispensable
role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two
Editedby:
homologs of intron less SbUSP gene which encodes for salt and osmotic responsive
ShabirHussainWani,
Sher-e-KashmirUniversity USP. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein.
ofAgriculturalSciences The role of the gene was functionally validated by developing transgenic tobacco and
andTechnology,India
compared with control [wild-type (WT) and vector control (VC)] plants under different
Reviewedby:
abioticstresscondition.Transgeniclines(T )exhibitedhigherchlorophyll,relativewater,
AnilKumarSingh, 1
IndianCouncilofAgricultural proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic
Research–IndianInstitute
potential, membrane stability, and lower electrolyte leakage and lipid peroxidation
ofAgriculturalBiotechnology,India
MariaAzucenaOrtega-Amaro, (malondialdehyde content) under stress treatments than control (WT and VC) plants.
InstitutoPotosinodeInvestigación Lower accumulation of H O and O− radicals was also detected in transgenic
CientíficayTecnológica,Mexico 2 2 2
lines compared to control plants under stress conditions. Present study confers that
*Correspondence:
overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup,
AvinashMishra
[email protected]; maintainsionhomeostasisandimprovesthephysiologicalstatusoftheplantundersalt
BhavanathJha
and osmotic stresses. Principal component analysis exhibited a statistical distinction
[email protected]
of plant response to salinity stress, and a significant response was observed for
Specialtysection: transgeniclinesunderstress,whichprovidesstressendurancetotheplant.Apossible
Thisarticlewassubmittedto
signaling role is proposed that some downstream genes may get activated by abiotic
PlantPhysiology,
asectionofthejournal stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative
FrontiersinPlantScience damages.Thestudyunveilsthatectopicexpressionofthegenemitigatessaltorosmotic
Received:14December2015 stressbyscavengingROSandmodulatingthephysiologicalprocessoftheplant.
Accepted:01April2016
Published:21April2016
Keywords:abioticstress,drought,halophyte,salinity,transgenic
Citation:
UdawatP,JhaRK,SinhaD,MishraA
andJhaB(2016)Overexpressionofa INTRODUCTION
CytosolicAbioticStressResponsive
UniversalStressProtein(SbUSP) Abiotic stresses, especially salinity and drought are an important limiting factor in plant
MitigatesSaltandOsmoticStress
growth due to increased salinization of soil and water as well as the global scarcity
inTransgenicTobaccoPlants.
of water resources (Vinocur and Altman, 2005). Abiotic stresses (salinity, dehydration,
Front.PlantSci.7:518.
doi:10.3389/fpls.2016.00518 extreme temperature, highlight intensity, etc.) pessimistically impinge on plant growth
FrontiersinPlantScience|www.frontiersin.org 1 April2016|Volume7|Article518
fpls-07-00518 April19,2016 Time:16:47 #2
Udawatetal. SbUSPProvidesAbioticStressTolerance
and development through biomass accumulation and grain This might be due to the inactivation of σ70 promoter at high
yield. Abiotic stresses are multigenic in nature, and the cellular temperature stress. USP homologs are ubiquitous in plants and
responsesareofteninterconnected.Thus,abioticstresstolerance encoded by gene families while the functions of USPs remain
in a plant is synchronized action of multiple stress responsive largely unknown (Loukehaich et al., 2012). It appears that USP
genes,whichcommunicateinstresssignaltransductionpathways boost up the rate of cell survival during prolonged exposure to
with other components (Tuteja, 2007). Salinity negatively stressagents,anditmayendowplantswithwiderangingstress
affects plant growth in two distinct phases; initial osmotic enduranceactivity(Zahuretal.,2009).TheUspAprotectscells
phase and the late ionic phase (Munns and Tester, 2008). fromdetrimentaleffectoftoxicchemicalsandosmoticstressor
Thesephasescanbeexperimentallydistinguishedbymeasuring UV light (Kerk et al., 2003). Few plant USP genes have been
effects in the short term (within minutes to a few days) characterizedviz.Gossypiumarboreum,Astragalussinicus,Vicia
upon addition of salt or after longer (several days to weeks) faba, Solanum pennellii, Barley, and Viridiplantae (Sauter et al.,
times (Yamamoto et al., 2015). The cellular and molecular 2002; Chou et al., 2007; Merkouropoulos et al., 2008; Maqbool
responsesincludeaccumulationofstressproteins,upregulation etal.,2009;Zahuretal.,2009;Lietal.,2010;Isokpehietal.,2011;
ofstressresponsivegenesforantioxidants,transcriptionfactors, Loukehaich et al., 2012). USPs are divided into two categories;
compatible solutes, transporters/channels, and phytohormones, first group binds with ATP (UspF and UspG-type) and second
etc. Molecular mechanism of stress responses plays a key do not, such as UspAs and UspA-like group (Tkaczuk et al.,
role in devising a strategy to engineer stress tolerant plants 2013). The number of copies of USPs varies amongst different
(Zahur et al., 2009). It is observed that abscisic acid (ABA) organismsandmajorityofthemhavemanyparalogousofUSPs.
accumulates under drought stress condition, which promotes For instance, the known copies of genes encoding USPs in
expression of numerous ABA dependent drought and abiotic A. thaliana are four, Nitrosomonas europaea and Archaeoglobus
stress responsive genes. Though substantial researches have fulgidus are six and eight, respectively (Tkaczuk et al., 2013).
been carried on the abiotic stress response of plants, there is Kvint et al. (2003) proposed that the ATP-binding Usp domain
inadequateinformationonmolecularregulatorymechanismsof is a member of a group of α/β domains called the HUP
environmental stress responsive genes and proteins (Isokpehi domain (HIGH-signature proteins, Usp-like domains, and PP-
etal.,2011).Thereareanumberofreportsonstressassociated ATPases).
genes and proteins, but they are still functionally unknown A halophyte Salicornia brachiata requires NaCl for tissue
or poorly characterized. Functional validation of these stress culture (Joshi et al., 2012), owns sulfur rich seed storage
responsive genes or proteins will unveil a new regulatory proteins(Jhaetal.,2012),uniqueoligosaccharides(Mishraetal.,
molecular mechanism, which may consider a promising 2013), and metabolites (Mishra et al., 2015). Several candidate
approach for developing crop plants, tolerant to environmental genesfromthishalophyteextensivelyexploredforabioticstress
stresses. responsive genes and promoters for developing abiotic stress
The universal stress proteins (Usp) were named initially as tolerant transgenic plants (Jha et al., 2011; Chaturvedi et al.,
C13.5proteinonthebasisoftheirmigrationontwo-dimensional 2012, 2014; Joshi et al., 2013; Singh et al., 2014a,b; Tiwari
IEF-PAGE gel. Nevertheless, UspA quickly became extensively et al., 2014, 2015, 2016; Udawat et al., 2014). The present
acknowledged,andtheproteinhasspecifieditsnametoarising study addresses first report on the functional characterization
orthologous group of proteins, the UspA superfamily (Kvint of the SbUSP gene by ectopic expression in a model plant and
etal.,2003).TheuniversalstressproteinA(UspA)superfamily further analyses under varying abiotic stress condition. It was
covers primordial and preserved group of proteins originated observed that overexpression of the SbUSP gene mitigates salt
in bacteria, archaea, fungi, flies, and plants. Kerk et al. (2003) and osmotic stress and provides endurance to the transgenic
reportedenigmaticfunctionsof USPAcontainingproteinkinases plantbymodulatingphysiologicalprocesses.
in Arabidopsis. Even though the regulation of the E. coli UspA
gene has been known to some extent, but the precise role of
MATERIALS AND METHODS
Usp proteins and Usp domains stay unfathomable, they have
been emerged to be linked for providing resistance to DNA
Genomic Organization of SbUSP Gene
damaging agents and respiratory uncouplers. In E. coli, the
USPs have been grouped into four classes according to their Plant(S.brachiata)genomicDNAwasextractedusingmodified
structural analysis and amino acid sequence; Class-I: UspA, CTAB (N-cetyl-N,N,N-trimethyl ammonium bromide) method
UspC, UspD; Class-II: UspF and UspG; Class-III and Class-IV: (Bubner et al., 2004) with minor modifications, quantified
TwoUspdomainsofUspE(Isokpehietal.,2011).Therearesix using ND-1000 spectrophotometer and qualitatively analyzed
familiesofUSP genesviz.UspA,UspC,UspD,UspE,UspF,and by agarose gel electrophoresis. The SbUSP gene was amplified
UspG,whereas,UspBisnotconsideredasabonafideUspfamily withprimerpairs(F:5(cid:48)-ATGGCAATGTCTGATAAACC-3(cid:48) and
protein (Kvint et al., 2003). The six USP genes of E. coli have R:5(cid:48)-TCAGTGCTTAATCTTAGGCTT-3(cid:48))usinggenomicDNA
different functions linked to motility, adhesion and oxidative astemplate,clonedintothepGEM-Teasyvectorandsequenced
stressresistance(Loukehaichetal.,2012).Itisobservedthatthe (M/sMacrogen,S.Korea).
majorityofUspgenesaremonocistronicallyexpressedfromσ70 The copy number of SbUSP gene was determined using
promoter,buthightemperature,ethanol,andtetracyclinestress Southern blot analysis. Genomic DNA (2 µg) was digested
reduce the expression level of Usp proteins (Kvint et al., 2003). with restriction enzymes (EcoR1 and BamH1) and transferred
FrontiersinPlantScience|www.frontiersin.org 2 April2016|Volume7|Article518
fpls-07-00518 April19,2016 Time:16:47 #3
Udawatetal. SbUSPProvidesAbioticStressTolerance
to Hybond N+ membrane (Amersham Pharmacia, UK) using via GUS activity (Jefferson, 1987) using β-glucuronidase
alkaline transfer buffer (0.4 N NaOH along with 1 M NaCl) reporter gene staining kit (Sigma, USA). The integration of
byupwardcapillarytransfer.Theblotwashybridizedovernight transgene and its copy number was determined by Southern
at 42◦C with PCR generated DIG-11-dUTP labeled SbUSP blot analysis following manufacturer instructions (Roche,
probe (F: 5(cid:48)-ATGGCAATGTCTGATAAACC-3(cid:48) and R: 5(cid:48)- Germany).
AGGAGGACAATGCGTGCTT-3(cid:48)) and signals were detected
on X-ray film by using CDP-Star as a chemiluminescent Overexpression Analysis of SbUSP Gene
substrate, as described by manufacturer instructions (Roche, Total RNA was extracted from transgenic, VC and WT plants
Germany). (abiotic stressed and unstressed), quantified and cDNA was
prepared using Superscript II RT (Invitrogen, USA). The semi-
Subcellular Localization quantitativereversetranscriptasePCR(RT-PCR)wasperformed
The gateway technology was used to generate translational thriceindependently(eachcontainingthreeplants)fortheSbUSP
fusion cassette of SbUSP along with RFP (red fluorescent geneandβ-tubulingene(F:5(cid:48)-GGAGTCACCGAGGCAGAG-3(cid:48)
protein). The SbUSP CDS was PCR amplified using and R: 5(cid:48)-ATCACATATCAGAAACCACAA-3(cid:48)) was used as an
accuPrime Pfx DNA polymerase along with SbUSPF (5(cid:48)- internalreference.Similarly,quantitativeRealTimePCR(qRT-
CACCATGGCAGAAGTTGATT-3(cid:48))andSbUSPR(5(cid:48)-TCAGTG PCR) was performed to study the relative fold expression of
CTTAATCTTAGGCTT-3(cid:48))primers.ThePCRproduct,amplified SbUSPgeneintransgeniclinesundersalinitystress.
with blunt ends was cloned into entry vector (pENTER/D-
Analyses of T Transgenic Plants
TOPO, Invitrogen, USA) and sequenced. Subsequently, 1
LR recombination reaction was executed between an attL- Exposed to Varying Abiotic Stresses
containing entry clone pENTER/D-TOPO-SbUSP vector TheT seeds(25seedsfromeachplant)ofSbUSPoverexpressing
1
and an attR-containing destination vector pSITE-4CA (RFP) transgenic lines (L7, L11, L14) along with VC and WT
using gateway LR Clonase II enzyme mix (Invitrogen, USA). plants were germinated on MS media supplemented with NaCl
Cloning was confirmed by PCR, and resulting expression (200 mM) or mannitol (300 mM) for three weeks under
cassette (RFP:SbUSP) and control vector (pSITE-4CA:RFP) controlled conditions, and percentage seed germination was
were transformed to onion epidermal cells using particle studied(Pandeyetal.,2015).Percentgerminationwasrepeated
bombardment with gene gun (PDS-1000/He Biolistic, Bio- threetimesandeachcontainedthreebiologicalreplicates.Equal
Rad, USA). Transformants were observed after incubation size of hygromycin (20 mgL−1) positive T seedlings were
1
in dark for 12 h for transient expression of RFP using an transferred to MS media supplemented with NaCl (200 mM)
epifluorescence microscope (Axio Imager, Carl Zeiss AG, or PEG (10%) and subsequently incubated for 21 days for root
Germany). length, shoot length, and ion content analyses. Additionally,
45 days old plantlets (transgenic lines, VC and WT) were
Genetic Transformation to Tobacco subjected to NaCl (200 mM), PEG (10%), ABA (20 µM) or
Plants and Molecular Confirmation SA (10 µM) stress for 24 h; thereafter samples were harvested.
The function of SbUSP gene in transgenic plants was validated
The SbUSP CDS was amplified using proofreading enzyme
through morphological and physio-biochemical and molecular
accuPrime Pfx DNA polymerase along with primer pairs
(F: 5(cid:48)-CTCGAGATGGCAATGTCTGATAAACC-3(cid:48) and R: 5(cid:48)- analyses viz. fresh weight, dry weight, osmotic potential,
GGTACCTCAGTGCTTAATCTTAGGCTT-3(cid:48))havingKpnIand membrane stability index, electrolyte leakage, chlorophyll
− −
estimation,O estimation,invivolocalizationofH O andO
XhoI sites, respectively. The SbUSP cDNA was cloned in 2 2 2 2
radicals,etc.
pCAMBIA1301 via an intermediate pRT101 plant expression
vector and recombinant pCAMBIA1301:35S:SbUSP vector was
Quantification of Osmotic Adjustment
mobilized into Agrobacterium tumefaciens strain LBA4404.
Tobacco (Nicotiana tabacum cv. Petit Havana) plants were The leaf segments of 45 days old stressed and unstressed
transformedbyleafdiskmethod(Horschetal.,1985)andshoot tobacco plants were harvested and incubated at −20◦C. After
budsdirectlygeneratedfromleafdisksweretransferredtoshoot overnightincubation,plantsampleswerethawedandcentrifuged
elongationandsubsequentlytorootingmediasupplementedwith at 13,000 rpm for 10 min. The cell sap extract was used
hygromycin(20mgL−1). to measure cellular osmotic potential using vapor pressure
Putative transgenic lines (T and T ) were screened by osmometer (VAPRO, Wescor Inc., USA). The experiment was
0 1
growing on hygromycin (20 mgL−1), and the presence of carried out three times, and each contained three biological
transgenes was confirmed by PCR amplification of uidA (F: replicates.
5(cid:48)-GATCGCGAAAACTGTGGAAT-3(cid:48) and R: 5(cid:48)-TGAGCGTC
GCAGAACATTAC-3(cid:48)) and SbUSP genes. The activity of Relative Water Content (RWC)
reportergeneβ-glucuronidaseintheleafsegmentoftransgenic Freshweightfromcontrol(WTandVC)andtransgenictobacco
(overexpressing SbUSP gene), VC (vector control; transgenic plantswasdetermined,andleafsampleswerefloatedovernight
plant transformed with pCAMBIA1301 vector only) and in deionised water. The turgid weight was determined, and all
WT (wild-type plant, non-transgenic) plants was visualized samples were dried at a constant temperature. Dry weight was
FrontiersinPlantScience|www.frontiersin.org 3 April2016|Volume7|Article518
fpls-07-00518 April19,2016 Time:16:47 #4
Udawatetal. SbUSPProvidesAbioticStressTolerance
measured, and percent relative water content (RWC, %) was young leaf samples were incubated in deionized water in
calculated using following equation. RWC was measured three closed vials and kept overnight on a rotary shaker. Thereafter,
times,eachcontainingthreebiologicalreplicates. electrical conductivity was measured (L) using conductivity
t
meter (SevenEasy, Mettler Toledo AG 8603, Switzerland).
RWC(%)=100× Freshweight−Dryweight Subsequently,leafsampleswereautoclavedat120◦Cfor15min,
Turgidweight−Dryweight further,theywerecooledto25◦C,andelectricalconductivity(L )
0
wasagaindetermined(Luttsetal.,1996).Thepercentelectrolyte
Senescence Assay and Estimation of
leakagewascalculatedusingfollowingformula:
Chlorophyll Content
Leaf disks from 45 days old control and transgenic tobacco
L
plants were punched out, and 11 leaf disks of similar size from EL(%)=100× t
L
each plant were floated in 1/2 MS (control) supplemented with 0
NaCl (100, 150, or 200 mM) or PEG (10%) for 8 days. Effects
The MSI and EL experiments were repeated three times with
of each treatment were gaged by visually analyzing phenotypic
threebiologicalreplicateseach.
changes amongst leaf segments. Furthermore, total chlorophyll
contents were isolated from leaf disks and determined as per
gram fresh weight of tissue (Inskeep and Bloom, 1985). The Quantification of Proline, H O , and Lipid
2 2
experiment was repeated three times with three biological
Peroxidation
replicateseach.
The free proline content and H O level in the stressed and
2 2
Superoxide Estimation and Cell Viability unstressed WT and transgenic tobacco plants were determined
using methods described by Bates et al. (1973) and He et al.
Test
(2005),respectively.Astandardcurvewaspreparedusingknown
ProductionofsuperoxideradicalsunderNaCl(200mM)orPEG
amountofprolineandH2O2, andabsorbancewasmeasuredat
(10%) stress was quantified using 2,3-bis-(2-methoxy-4-nitro-
520and560nmfordeterminingfreeprolineandH O content,
2 2
5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay
respectively. Lipid peroxidation was analyzed by quantifying
(Hema et al., 2014). Leaf segments (of equal size and weight)
the amount of MDA (malondialdehyde) generated by TBA
wereincubatedfor 5hinpotassiumphosphatebuffer (20mM,
(thiobarbituric acid) reaction (Draper and Hadley, 1990). All
pH6.0)containing500µMXTTandincreaseinabsorbancewas
experiments were repeated three times with three biological
recordedat470nm.
replicateseach.
Similarly, leaf segments (similar weight and size) of 45 days
old stressed and unstressed plants were used for estimation
of cell viability by 2,3,5-triphenyltetrazolium chloride (TTC) Quantification of Total Soluble Sugar,
assay (Hema et al., 2014) in sodium phosphate buffer (pH Reducing Sugar, Starch, Total Amino
7.4). Leaf segments were thoroughly washed with sterile
Acid, and Polyphenol Content
water and incubated in TTC solution at room temperature
The total soluble sugar, reducing sugar and starch contents
in the dark for 6 h. Samples were boiled in 5 ml of 2-
weredeterminedagainstastandardcurvepreparedwithglucose
methoxy ethanol till aridness to dig-out bound formazan.
(Sigma–Aldrich, USA). Stressed and unstressed control and
Subsequently, 5 ml of 2-methoxy ethanol was again added, and
transgenic leaf samples were homogenized in 85% ethanol and
absorbance was recorded at 485 nm. Both experiments were
treated with anthrone reagent. Samples were boiled, cooled
carried out three times and each contained three biological
up to 25◦C, absorbance was measured at 620 nm and total
replicates.
solublesugarwascalculatedfromthestandardcurve.Reducing
Membrane Stability Index (MSI) and sugar was quantified by a colorimetric test using DNS method
(Yemm and Willis, 1954). An aliquot of glucose or leaf
Electrolyte Leakage (EL)
sample was mixed with 3 ml of DNS reagent, boiled, cooled
Healthyyoungleaveswerecollectedfromstressedandunstressed
to room temperature, and color intensity was measured at
WTandtransgenictobaccoplants,andMSIwascalculatedbythe
540 nm. For starch estimation, homogenized samples were
methoddescribedbySairam(1994).Leafsegmentswereplaced
treated with 52% perchloric acid, centrifuged, the supernatant
in sterile water, and one set (L ) was incubated at 40◦C for
1 was treated with 4 ml of anthrone reagent and absorbance
30minwhereasotherset(L )wasincubatedat100◦Cfor10min.
2 was measured at 630 nm (Ghose, 1987). Free amino acids
Electricalconductivityofsamplesfrombothsetswasmeasured,
and polyphenol contents were determined against a standard
andMSIwascalculatedusingfollowingformula:
curvepreparedwithglycineandcatecholsolution,respectively.
The ethanolic extract of samples was redissolved in ninhydrin
L
MSI=100×1− 1 reagent or folin–ciocalteu reagent, and absorbance (color
L
2
intensity) was measured at 570 or 650 nm, respectively. All
For electrolyte leakage, leaf samples were initially washed with experiments were carried out three times with three biological
sterile water to eliminate surface stacked electrolytes. Healthy replicateseach.
FrontiersinPlantScience|www.frontiersin.org 4 April2016|Volume7|Article518
fpls-07-00518 April19,2016 Time:16:47 #5
Udawatetal. SbUSPProvidesAbioticStressTolerance
In Vivo Localization of H O and O− and physio-biochemical measurements were taken as variables.
2 2 2
ObservationandvariabledatasetwereusedtogeneratePearson’s
Histochemical staining with 3,3-diaminobenzidine (DAB) or
correlation matrix, and PCA was analyzed using different
nitro-blue tetrazolium (NBT) was performed for in vivo
− softwareSigmaPlot(version12),SYSTAT(version13)andOrigin
analyses of H O and O radical production (Shi et al.,
2010). A meas2ur2e of 1 m2g ml−1 each stain solution was (version15).
prepared in phosphate buffer (10 mM pH 7.8 for NBT
and pH 7.3 for DAB). Leaf samples were immersed in the
RESULTS
respective staining solution in the dark for 2 h, thereafter
exposed to white light until blue and brown spots appeared.
In Vivo Localization and Genome
Total chlorophyll content was bleached and leaf samples were
documented. Organization
The in vivo subcellular localization study was accomplished by
Analyses of Ion Content transient expression analysis of translational fusion construct
Seedlings from stressed and unstressed, control and transgenic pSITE-4CA:SbUSP and vector pSITE-4CA:RFP (Figures 1A,B)
plants were subjected to dryness at 70◦C for 48 h and expressing RFP only in onion epidermal cells. The expression
dry weight was determined. Samples were acid digested with pSITE-4CA vector showed uniform red fluorescence,
overnight in perchloric acid-nitric acid solution (3:1) heated whereas RFP:SbUSP fusion construct revealed fluorescence
to dryness and redissolved in sterile water. Ion content was localized to the cytosolic membrane (Figure 1B). DAPI (4(cid:48),6-
measured using inductively coupled plasma optical emission diamidino-2-phenylindole)isafluorescentstain,whichbindsto
spectrometer (Optima2000DV, PerkinElmer, Germany). ICP double strand DNA at A-T rich region. DAPI has an emission
analysis was done three times with three biological replicates maximum at 461 nm, and in fluorescence microscopy, it is
each. detected through a blue filter. In the in vivo localization study,
blue spot of DAPI indicates the position of the nucleus (as
Quantitative RT PCR Analysis it binds with dsDNA). Overlapping of DAPI blue spots was
not observed with RFP spots in the cells, transformed with
The cDNA of all control and treated plant samples (WT,
RFP:SbUSP fusion construct. In the merged image, SbUSP
VC, and transgenic lines) viz. NaCl, PEG, ABA, and SA
was synthesized from 5 µg total RNA using Superscript bound RFP shows red fluorescence in the cytosol, outside
the nucleus and inner side of the membrane. The result
RT III first strand cDNA synthesis kit (Invitrogen, USA).
suggested that SbUSP is a membrane bound cytosol-localized
The resulting cDNAs were subjected to real time qRT PCR
protein. The in silico analysis also confirmed that SbUSP is
analyses for selected antioxidant enzyme encoding genes of
host plant (NtSOD, F: 5(cid:48)-AGCTACATGACGCCATTTCC-3(cid:48) membrane bound cytosolic protein. The SbUSP ORF amplified
and R: 5(cid:48)-CCCTGTAAAGCAGCACCTTC-3(cid:48); NtAPX, F: 5(cid:48)- using full length gene specific primers from cDNA, and
CAAATGTAAGAGGAAACTCAGAGGA-3(cid:48) and R: 5(cid:48)-CAG genomic DNA showed similar size, 486 bp. The comparative
CCTTGAGCCTCATGGTACCG-3(cid:48); NtCAT, F: 5(cid:48)-AGGTAC sequence analysis of cDNA clone compared to genomic clone
CGCTCATTCACACC-3(cid:48) and R: 5(cid:48)-AAGCAAGCTTTTGAC further reveals single exon structure and confirms intron-less
CCAGA-3(cid:48)) followed by melt curve analysis for the validation genomic organization the gene (data not shown). Furthermore,
Southern blot analysis was commenced to identify the number
of specificity of reaction. The relative fold expression change
of SbUSP copies (homologs) in the genome of S. brachiata.
was calculated using the CT method, and internal reference
gene β-tubulin was used as a reference (Livak and Schmittgen, The analysis revealed a double copy of the SbUSP gene as
the probe hybridized with two fragments of genomic DNA
2001).
(Figure1C).
Statistical Analysis
Molecular Analyses of Transgenic
For each set of experiment, data from three replicates, each
Tobacco Plants
containing three biological replicates were documented. Data
sets were articulated as mean ± SE and were used to HealthyleavesfromtheSbUSPgene(Figure2A)overexpressing
determine the significance of difference by analysis of variance T lines (L1 to L17 lines), WT and VC plants were harvested
1
(ANOVA) amongst the means of WT, VC, and transgenic and screened for the presence of transgenes (Supplementary
plantsofeverytreatmentset.Forcomparisonofmultiplemeans Figure S1). Furthermore, Southern blot analysis confirmed
Tukey HSD was used, p < 0.05 was considered significantly presence of single copy transgene amongst L4, L7, L8, L11,
different from each other and designated by different letters. L12, and L14 transgenic lines (Supplementary Figure S2A).
All dataset was analyzed individually and in combination The SbUSP gene overexpressing transgenic lines exhibited a
by principal component analysis (PCA) and respective heat high level of gus expression along with VC plants (Figure 2B).
maps were generated. PCA is a multivariate analysis method ComparativeectopicexpressionoftheSbUSPgenewasobserved
by which correlation between variables were studied among by semi-quantitative RT-PCR and quantitative Real Time PCR
multidimensional datasets. Plants grown under varying stress (qRT-PCR) for transgenic lines (Supplementary Figure S2)
were selected as observations, whereas different morphological using β-tubulin as an internal reference gene. The analysis
FrontiersinPlantScience|www.frontiersin.org 5 April2016|Volume7|Article518
fpls-07-00518 April19,2016 Time:16:47 #6
Udawatetal. SbUSPProvidesAbioticStressTolerance
FIGURE1|Sub-cellularlocalizationofSbUSPproteinandgenomeorganization.(A)VectorpSITE-4CA(RFP)andSbUSP-1:RFPgeneconstruct,
(B)TransientexpressionofRFPaloneandSbUSP:RFPtranslationalfusionproteinontransformedonionepidermalcellsand(C)SouthernhybridizationofSalicornia
brachiatagenometodeterminethecopynumberofSbUSPgene.
FIGURE2|GenetictransformationandHistochemicalGUSassayoftransgenictobaccoplants.(A)SchematicrepresentationofSbUSP-pCAMBIA1301
planttransformationvectorconstructand(B)HistochemicalGUSassayoftransgeniclines(L7,L11,andL14)alongwithcontrolplants(WTandVC).
revealed high expression of the SbUSP gene in transgenic lines that transgenic lines (L7, L11, and L14) showed significantly
L7, L11, and L14, therefore, selected for further functional (p < 0.05) higher percent seed germination compared to
validationbymorphological,physio-biochemicalandmolecular control plants (WT and VC) under the stressed condition.
analyses. Similarly,growthparameters,includingfreshweight,dryweight,
shoot and root length were significantly (p < 0.05) improved
in selected transgenic lines (L7, L11, and L14) compared to
Overexpression of the SbUSP Gene
controlplants(WTandVC)understressconditions(Figure4).
Enhances Plant Growth under Salt and
Leaf segments reduce their viability and undergo senescence
Osmotic Stress under salt and osmotic stress (Figure 5A). Salt and osmotic
Percentage seed germination was determined under control, stress induced damages were lower in SbUSP overexpressing
NaCl, and osmotic stress condition (Figure3). It was observed transgenic lines compared to their counterparts (WT and
FrontiersinPlantScience|www.frontiersin.org 6 April2016|Volume7|Article518
fpls-07-00518 April19,2016 Time:16:47 #7
Udawatetal. SbUSPProvidesAbioticStressTolerance
FIGURE3|Seedgerminationanalysisoftransgenictobaccoplantsunderabioticstress.(A)Morphologyand(B)percentseedgerminationanalysisof
transgeniclines(L7,L11,andL14),WTandVCplantsundersalinityandosmoticstresscondition.Barsrepresentmeans±SEandvalueswithdifferentlettersare
significantatP<0.05.
FrontiersinPlantScience|www.frontiersin.org 7 April2016|Volume7|Article518
fpls-07-00518 April19,2016 Time:16:47 #8
Udawatetal. SbUSPProvidesAbioticStressTolerance
FIGURE4|Plantgrowthanalysesundersaltandosmoticstress.Comparativestudyof(A)morphology,(B)freshweight,(C)dryweight,(D)rootlength,and
(E)shootlengthoftransgeniclines(L7,L11,andL14)andcontrolplants(WTandVC)undersaltandosmoticstress.Barsrepresentmeans±SEandvalueswith
differentlettersaresignificantatP<0.05.
VC). Transgenic lines exhibited reduced rate of necrosis and stress tolerance. Abiotic stress also leads to the generation of
therefore,significantly(p<0.05)higheramountofchlorophyll superoxide radicals. It was demonstrated by the reduction of
content was observed in transgenic plants related to WT and tetrazolium salts XTT, resulting significant (p < 0.05) increase
VC plants. A degradation in cellular chlorophyll content was inabsorbance(470nm)intransgeniclinescomparedtocontrol
observed in WT and VC plants compared to transgenic lines plants (WT and VC) under stress condition (Supplementary
(Figure 5B). Under control condition, chlorophyll content of FigureS3).
WT,VC,andtransgenicplantswascomparable,whereasunder ProlineandMDAcontentswerecomparableinWT,VC,and
increasing salt and osmotic stress, significantly higher amount transgeniclinesundercontrolcondition(Figure7).Transgenic
of chlorophyll was found in transgenic lines. Additionally, linesaccumulatedsignificantly(p<0.05)highprolinecompared
transgenic lines showed significantly (p < 0.05) enhanced cell tocontrolplantsunderstress(Figure7A).Incontrast,elevated
viabilitycomparedtoWTandVCplantsunderstresscondition H O and MDA contents (p < 0.05) were found in control
2 2
(Figure5C). plants under stress condition compared to transgenic lines
(Figures 7B,C). Results further confirmed its (SbUSP) role in
SbUSP Overexpression Alleviates ROS oxidativestresstolerance.
Buildup under Salinity and Oxidative
Overexpression of the SbUSP Gene
Stress
Improves Physiological Status of Plant
The WT and VC plant leaves exhibited deeper NBT and DAB
under Stress
stainingthantransgeniclinesexposedtosaltandosmoticstress
(Figure6).Resultsconfirmedthatcontrolplants(WTandVC) Transgenic,WTandVCplantsshowedalikephysiologicalstatus
−
accumulate more O and H O compared to transgenic plants regarding RWC, EL, MSI, and OP under control condition
2 2 2
andthusvalidatingtheroleofSbUSPgeneinprovidingoxidative (Figure 8). However, transgenic lines showed significantly
FrontiersinPlantScience|www.frontiersin.org 8 April2016|Volume7|Article518
fpls-07-00518 April19,2016 Time:16:47 #9
Udawatetal. SbUSPProvidesAbioticStressTolerance
FIGURE5|Leafdiskassay,estimationofchlorophyllcontentandcellviability.(A)Leafdiskassay,(B)totalchlorophyllcontent,and(C)cellviabilityof
transgenic(L7,L11,andL14)andcontrolplants(WTandVC)undersaltandosmoticstresscondition.Barsrepresentmeans±SEandvalueswithdifferentletters
aresignificantatP<0.05.
(p < 0.05) higher RWC and MSI under stress condition acid and polyphenol content decreased under stress condition
compared to control plants. Furthermore, transgenic plants compared to the control condition, irrespective of plants. But,
exhibited considerably (p < 0.05) reduced electrolyte leakage transgenic lines accumulated significantly (p < 0.05) higher
and osmotic potential under stress condition. Transgenic lines content of reducing sugar, free amino acid and polyphenol
exhibitedbetterosmoticadjustmentandthusphysiologicalstatus compared to control plants (WT and VC) under stress
oftransgenicplantsimproves. condition (Figures 9B–D). Results showed the accumulation
of primary metabolites/osmolytes in transgenic plants, which
improve the performance of transgenic plants under stress
Total Soluble Sugar, Reducing Sugar,
condition.
Free Amino Acids, and Polyphenol
Contents Increase in the SbUSP Overexpression of the SbUSP Maintains
Overexpressing Lines Ion Homeostasis
Total soluble sugar and reducing sugar were found almost For analyses of ion content, the WT, VC, and SbUSP
similarinWT,VC,andtransgeniclinesundercontrolconditions overexpressing transgenic seedlings were grown on MS media
(Figure 9). A significant difference (p < 0.05) was observed supplementedwithNaCl(200mM)andgrownfor28days.The
amongst WT, VC, and transgenic plants exposed to salt and Na+ andK+ contentinwholeseedlingsweremeasuredinWT,
osmoticstress.Stresshadnoeffectontotalsolublesugarcontent VC, and transgenic seedlings exposed to salt stress. Potassium
in WT and VC plants, whereas transgenic lines accumulate ion content was increased in transgenic lines compared
significantly (p < 0.05) high content compared to control to control plants and control conditions (Supplementary
plants as well as the control condition (Figure 9A). The Figure S4). In contrast, Na+ accumulation was observed in
transgeniclineL7showedfivefold,whereasL11andL14showed control plants (WT and VC) compared to transgenic lines
a sixfold increase of soluble sugar content under salt stress. under salt stress. The K+/Na+ ratio, an indicator of ion
Similarly, under osmotic stress, line L7 showed more than homeostasis was almost similar in all plants under control
sixfoldincreaseandlineL11andL14showedeightfoldincreases condition. The ratio decreased in control plants (WT and
in total soluble sugar content. Reducing sugar, free amino VC) under salt stress, whereas elevated ratio was detected
FrontiersinPlantScience|www.frontiersin.org 9 April2016|Volume7|Article518
fpls-07-00518 April19,2016 Time:16:47 #10
Udawatetal. SbUSPProvidesAbioticStressTolerance
FIGURE6|Invivolocalizationofperoxideandsuperoxidefreeradicals.(A)DABand(B)NBTstainingoftransgenic(L7,L11,andL14)andcontrol(WTand
VC)leaves.
The SbUSP Gene May Also Involve in
in transgenic lines compared to control plants and control
condition (Supplementary Figure S4). The high K+/Na+ ratio Transcriptional Regulation of Antioxidant
indicates that transgenic lines improved their ion homeostasis Enzyme-Encoding Genes
under salinity and thus performed better and showed stress Transcript expression of ROS scavenging enzyme genes viz.
endurance. superoxidedismutase(NtSOD),catalase(NtCAT),andascorbate
FrontiersinPlantScience|www.frontiersin.org 10 April2016|Volume7|Article518