Research Article
Leaf Rolling and Stem Fasciation in Grass Pea (
Lathyrus sativus
L.) Mutant Are Mediated through Glutathione-DependentCellular and Metabolic Changes and Associated with a MetabolicDiversion through Cysteine during Phenotypic Reversal
Dibyendu Talukdar
1
and Tulika Talukdar
2
Department of Botany, R.P.M College, University of Calcutta, Uttarpara, Hooghly, West Bengal , India
Department of Botany, Acharya Prafulla Chandra Roy Government College, University of North Bengal, Darjeeling,West Bengal , India
Correspondence should be addressed to Dibyendu alukdar; dibyendutalukdar@gmail.comReceived February ; Accepted April ; Published May Academic Editor: Pavel Hozak Copyright © D. alukdar and . alukdar. Tis is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.A
Lathyrus sativus
L. mutant isolated in ethylmethane sulonate-treated M
2
progeny o mother variety BioL- and designatedas
rlfL-
was characterized by inwardly rolled-lea and stem and bud asciations. Te mutant exhibited karyomorphologicalpeculiaritiesinbothmitosisandmeiosiswithoriginoaneuploidy.Temitosiswasvigorouswithhighrequencyodivisionalcellsandtheirquickturnoverpresumablysteeredcellprolierations.Signicanttranscriptionalupregulationsocysteineandglutathionesynthesis and concomitant stimulations o glutathione-mediated antioxidant deense helped
rlfL-
mutant to maintain balancedreactiveoxygenspecies(ROS)metabolisms,asdeducedbyROS-imagingstudy.Glutathionesynthesiswasshutdowninbuthioninesuloximine- (BSO-) treated mother plant and mutant, and lea-rolling and stems/buds asciations in the mutant were reversed,accompanied by normalization o mitotic cell division process. Antioxidant deense was downregulated under low glutathione-redox but cysteine-desulurations and photorespiratory glycolate oxidase transcripts were markedly overexpressed, preventingcysteine overaccumulation but resulted in excess H
2
O
2
in BSO-treated mutant. Tis led to oxidative damage in prolieratingcells, maniested by severe necrosis in rolled-lea and asciated stems. Results indicated vital role o glutathione in maintainingabnormal prolierations in plant organs, and its deciency triggered phenotypic reversal through metabolic diversions o cysteineand concomitant cellular and metabolic modulations.
1. Introduction
Intracellular thiol redox status is a critical parameter indetermining plant growth and development in response tocontinuous production o reactive oxygen species (ROS)[]. In aerobic cells, ROS homeostasis is delicately balanced[, ] and mediates crucial intracellular signaling pathways
which are essential or cell survival []. However, an excesso ROS ormation shifs the redox balance in avor o oxidative state which leads to cell damage and death. Teincrease o ROS, however, can induce an adaptive response,consisting o a compensatory upregulation o antioxidantsystems, aimed to restore the redox homeostasis [, ].
Glutathione (reduced orm: GSH; oxidized orm: GSSG),the tripeptide
-glutamylcysteinylglycine, is the principalantioxidant component and predominant orm (
>
%) o nonprotein thiol within a cell. In plants, GSH is synthesizedenzymatically rom its constituent amino acids via two-stepAP-dependent pathway catalyzed by
-glutamylcysteinesynthetase (
-ECS), the rate-limiting step, and can be inhib-ited by treating with BSO (L-buthionine suloximine) andGSH synthetase (GSHS) []. Owing to the presence o astrong nucleophilic thiol group on its cysteine (Cys) residue,GSH interacts with numerous cellular components as anefficient redox buffer, provides valuable inormation on cel-lular redox state, and, most importantly, regulates enzymaticprocesses both upstream o thiol-cascade and downstreamo thiol metabolites in order to maintain plant growth and
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 479180, 21 pageshttp://dx.doi.org/10.1155/2014/479180
BioMed Research Internationalmaximize cellular deenses against stresses [, ]. GSH is
an important actor controlling cell prolierations duringtumour growth in animal cells []. Elevated GSH levelswere observed in various types o prolierative response andprogression o cell cycle []. Moreover, the content o GSH insome tumor cells is typically associated with higher levels o GSH-related enzymes, such as
-ECS activities []. In plants,GSH is crucial in postembryonic development o the root[] and the shoot [], but despite many morphological and
biochemical eatures similar to those ound in animal cells,evidence or thiol involvement in plant tumorigenesis is notconvincing []. GSH plays central role in regeneration o reduced ascorbate rom its oxidized orm dehydroascorbate(DHA). Te resultant glutathione disulde or GSSG is thenrecycledtoGSHbyNADPH-dependentactionoglutathionereductase (GR) in ascorbate-GSH cycle []. Te reducedascorbate is used as an exclusive coactor by ascorbateperoxidase during scavenging o H
2
O
2
[]. Te H
2
O
2
isone o the prominent diffusible ROS within the cell and iscontinuously generated in aerobic cells by photorespiration,activity o superoxide dismutase (SOD), and other reasons.Dual roles o H
2
O
2
as an inducer o oxidative stress and apowerul signaling molecule or antioxidant upregulationshave been investigated in crop plants, experiencing bioticand abiotic stresses [–]. Te conjugation o GSH to
heavy metals, herbicides, and xenobiotics is accomplished by multiunctional enzymes glutathione
S
-transerases (GSs)[], establishing the now widely accepted role o GSs as cellhousekeepers involved in the detoxication o endogenousas well as exogenous substances. More recently, several GSisoenzymes have been shown to modulate cell signalingpathways that control animal cell prolieration and cell death(apoptosis) [, ]. Blockage o the GSH/GS detoxication
system enhanced the chemosensitivity o several tumor celllines. Consequently, excess generation o oxidative inter-mediates and depletion o GSH have been ound to eitherprecede the onset o apoptosis or render the cells moresensitiveto death[, ]. Glutathioneperoxidases (GPXs) are
alsoinvolvedinremovaloorganicperoxidesandH
2
O
2
usingGSHorthioredoxinsaselectrondonors[].TeGSH/(GSH+GSSG) ratio, considered as GSH redox state, is likely to be armore inuential in the antioxidant deense, controlling geneexpressionsandthionylationprocessthantheabsolutesizeo the glutathione pool [].Cysteine (Cys), oneo the vitalbuilding blocks o GSH, isthe rst stable and committed molecule in plant metabolismthat contains both sulur and nitrogen, synthesized by theO-acetylserine (thiol)lyase (OAS-L) either in ree activehomodimer or in association with serine acetyltranserase(SA)asaninactivesubunitothecysteinesynthasecomplex[, ]. OAS-L is the most enigmatic enzymatic systems
in sulur metabolisms and its isoorms along with SA indifferent cellular compartments are under strict regulationsin plants responding to environmental stresses [, , ].
While depleting Cys level escalates the stress sensitivity [,], ree Cys, but not GSH, above a certain concentrationthreshold reportedly has the ability to exacerbate the proox-idant properties due to high reactivity o its thiol moiety [, ]. Te opposite mechanisms o Cys biosynthesis and its
cellular degradation have immense signicance in maintain-ingCyshomeostasisandthisisundertightregulationsunderstressul environments [–]. Studies indicate that Cys
degradation is mainly mediated through the desulurationo L-Cys and D-Cys by L-cysteine desulydrase (LCD) andD-cysteine desulydrase (DCD), respectively, rather thanpromoting Cys biosynthesis in high plants []. Te resultanthydrogen sulde participates in different physiological activ-ities to promote plant growth processes but can also induceoxidative stress to promote cell death [–].
Development o plant organ is highly dynamic andinvolves the controlled initiation o new primordia romgroups o stem cells called meristems. Leaves and the axillary meristems that generate branches and owers are initiatedin regular patterns under tight cellular regulations rom theshoot apical meristem (SAM), balancing cell prolierationwith the incorporation o cells into new primordia [].Tis balance is essential to maintain SAM integrity and thestereotypical pattern o organ initiation throughout develop-ment. Lea rolling and stem prolieration are two importantphenomena in plant developmental process [–]; while
lea rolling is supposed to be an adaptation to droughttolerance mechanisms, prolierative growth in stems, buds,and in other plant organs ofen leads to asciation throughdisturbances in SAM. Although moderate lea rolling andasciationinplant partshave longbeen utilizedorcropyieldenhancement [], they are still regarded as an enigmaticprocess as nothing is known regarding intrinsic cellular andbiochemical processes involved in cell prolierations duringrolling and asciation. Most o the studies in shoot meristemprolieration have come rom mutations in
Arabidopsis
suchas
clavata
,
mgoun
and
mgoun
,
fasciata
and
fasciata
, and
fully fasciated
[] and in maize [], revealing involvement
o genetic events in the process. o date, rice mutantswith rolled leaves have been isolated []. However, despiteimmense importance o lea rolling and stem asciation incell physiological and agronomic point o view and roles o GSH-redoxstatusinregulationoplantgrowthevents,scanty work has been done in this direction []. Also, no reportsare available regarding cytogenetic behavior o prolierative vegetative and reproductive cells in plants. Grass pea is a very hardy cool-season legume crop, used both or oodand eed worldwide. Te novelty o this crop in cell biology researchhasbeenmaniestedthroughdevelopmentorobustcytogenetic and mutation stocks to decipher the cellular andmetabolic events leading to plant growth, development, andstress tolerance [–]. Here, we isolated one unique grass
pea mutant, exhibiting characteristic lea-rolling and stem aswellasaxillarybudasciation,inM
2
progenyoethylmethanesulonate- (EMS-) mutagenized population. Te mutantwas advanced to next generation through sel-pollinationto perorm a detail study. Te main goal o the presentinvestigation was to trace the intrinsic cellular and GSH-mediated mechanisms behind the phenotypic abnormalitiesin the mutant plants with the objectives to () measurethe Cys and GSH pool, () investigate the activities andmRNA gene expressions o Cys and GSH biosynthesis andGSH-dependent antioxidant systems, () assess the mitoticdelity and chromosomal anomalies, and () detect the ROS
BioMed Research International accumulation and oxidative damage in untreated and BSO-treated tissues o mother variety and mutant line.
2. Materials and Methods
.. Induction and Detection of Mutants.
Fresh seeds o grasspea (
Lathyrus sativus
L. cv. BioL-) were collected romPulses and Oilseed Research Station, Berhampore, WestBengal, India, and grown or two seasons ( and )in a private arm at Kalyani (
∘
N/
∘
E), West Bengal,India.Aferascertaininguniormityoseedageandhomozy-gosity,reshseedspresoakedinwaterorhweretreatedwithreshly prepared .%, .%, and .% aqueous solutiono ethylmethane sulonate (EMS) or h with intermittentshaking at
25 ± 2
∘
C keeping a control (distilled water). Aferthe stipulated period, seeds were thoroughly washed withrunning tap water and sown in the eld treatment wise (seeds treatment
−1
) along with untreated seeds as control intriplicate during November (temperature
∘
C/
∘
C,day/night, relative humidity
72 ± 4
%, photoperiod h, ir-radiance –
mol m
−2
s
−1
). Seled seeds o individualM
1
plants were harvested separately and were grown inthe next season in a randomized block design keeping adistance o cm between rows and cm between plantsto raise M
2
progeny. Standard agronomic practices wereollowed to grow healthy plant progeny. Phenotypes o about M
2
individuals were screened during winter o and . One plant with characteristic in-rolled leaets andprolierationinstemandaxillarybudshasbeenisolatedinM
2
progeny o EMS- (.%, h) treated population. Seeds (seeds)othisvariantplantwerecareullyharvestedandsowninthenextseasontoraiseM
3
progeny.Temutantphenotypewastruebreeding,andbasedonmorphologicalpeculiarities,the mutant was designated as
rlfL-
(
Rolled leaet and fasci-ated Lathyrus mutant-
). Further biochemical and molecularcharacterizations o the mutant were perormed on theseprogeny plants.
.. Culture Conditions and BSO reatment Protocol.
Grasspea seeds obtained rom M
3
plants o
rlfL-
mutant and itsmother variety BioL- were surace-sterilized with %ethanol or min, rinsed twice in deionized water, and thenplaced on water-moistened lter papers at dark to germinateat
∘
C. Germinated seedlings were immediately placedin polythene pots ( plants pots
−1
) containing mL o Hoagland’s number nutrient media and were permittedto grow or d. Te media were either unsupplemented(control plants) or supplemented (three treatment sets)with mM BSO (L-buthionine suloximine, Sigma-Aldrich,Bangalore, India) and the seedlings were allowed to grow or another d. Mother variety and
rlfL-
plants submittedto untreated (mM BSO) condition were used as mothercontrol (MC) and mutant control (MuC), respectively, whilethose treated with BSO were designated as treated mother(M) and treated mutant (Mu), respectively. Plants wereharvested at d growth period. Te experiment was carriedout in a completely randomized block design with threereplicatesinanenvironmentallycontrolledgrowingchamberunder a -hour photoperiod, temperature o /
18 ± 2
∘
C,relative humidity o
70 ± 2
%, and a photon ux density o
molm
−2
s
−1
. Shoots and roots were rinsed thoroughly with sterile distilled water and oven-dried at
∘
C or h toweigh dry mass.
.. Estimation of Glutathione, Cys, and Assay of Tiol Metab-olizing Enzymes.
Reduced and oxidized orms o glutathionewere measured ollowing the methods o Griffith []. Forenzyme assay, plant tissue (leaves and stems) was homog-enized in buffers specic or each enzyme under chilledconditions. Te homogenate was squeezed through ourlayers o cheese cloth and centriuged at ,g or minat
∘
C. Te protein content o the supernatant was measuredusing BSA as standard []. Assay o serine acetyltrans-erase (SA; EC ...) activity was perormed ollowingBlaszczyk et al. []. An enzyme unit was considered asthe amount o enzyme catalyzing the acetylation o pmolo L-serine per minute. Te OAS-L (EC ...) activity was assayed by measuring the production o L-Cys. Assay was started by the addition o
L crude extract (
g
L
−1
total protein in mM phosphate buffer, pH .).Reactions were conducted in mM phosphate buffer (pH.) in the presence o mM dithiotreitol (D), .mMO-acetyl L-serine (OAS), and mM sodium sulde (Na
2
S)in a total volume o
L assay mixture and allowed toproceed or min at
∘
C. Te reaction was terminatedby the addition o .mL o .% trichloroacetic acid [].Cys content was measured spectrophotometrically (Perkin-Elmer, Lambda , Mumbai, India) at nm ollowingGaitonde []. A blank control with all compounds exceptOAS was maintained, and the linearity o the assay waschecked with . and
L o added crude lea extract. Assay o
-ECS (EC ...) and LCD (EC ...) was done by ollowing Seelig and Meister [] and Bloem et al. [],
respectively. LCD (EC ...) activity was measured by thereleaseosulderomCysinatotalvolumeomLconsistingo .mM dithiothreitol, .mM L-Cys, mM RIS/HCl,pH ., and enzyme extract []. Te reaction was initiatedby the addition o L-Cys. Afer incubation or min at
∘
Cthereactionwasterminatedbyadding
LomMFeCl
3
dissolved in .NHCl and
L o mM N,N-dimethyl-p-phenylenediamine dihydrochloride dissolved in .NHCl[]. Te ormation o methylene blue was determined atnm.SolutionswithdifferentconcentrationsoNa
2
SwerepreparedandtreatedinthesamewayastheassaysamplesandwereusedorthequanticationoenzymaticallyormedH
2
S.DCD (EC ...) activity was determined in the same way,but D-Cys was used instead o L-Cys []. Four samples pertreatment were collected with our replications in the assays.
.. Assay of Antioxidant Enzymes.
issue o mg washomogenized in mL o mM K-phosphate buffer (pH .)containing mM EDA, mM D, and % (w/v) polyvinylpyrrolidone using a chilled mortar and pestle kept in an icebath. Te homogenate was centriuged at ,g at
∘
C ormin. Clear supernatant was used or enzyme assays. Formeasuring APX activity, the tissue was separately ground in
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