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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 sulonate-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 karyomorphologicalpeculiaritiesinbothmitosisandmeiosiswithoriginoaneuploidy.Temitosiswasvigorouswithhighrequencyodivisionalcellsandtheirquickturnoverpresumablysteeredcellprolierations.Signi󿬁canttranscriptionalupregulationsocysteineandglutathionesynthesis and concomitant stimulations o glutathione-mediated antioxidant deense helped
 rlfL-󰀱
 mutant to maintain balancedreactiveoxygenspecies(ROS)metabolisms,asdeducedbyROS-imagingstudy.Glutathionesynthesiswasshutdowninbuthioninesuloximine- (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 deense was downregulated under low glutathione-redox but cysteine-desulurations 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 prolieratingcells, maniested by severe necrosis in rolled-lea and asciated stems. Results indicated vital role o glutathione in maintainingabnormal prolierations in plant organs, and its de󿬁ciency 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-stepAP-dependent pathway catalyzed by 
 
-glutamylcysteinesynthetase (
-ECS), the rate-limiting step, and can be inhib-ited by treating with BSO (L-buthionine suloximine) 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 inormation 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 deenses against stresses [󰀱, 󰀳]. GSH is an important actor controlling cell prolierations duringtumour growth in animal cells [󰀵]. Elevated GSH levelswere observed in various types o prolierative 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 disul󿬁de or GSSG is thenrecycledtoGSHbyNADPH-dependentactionoglutathionereductase (GR) in ascorbate-GSH cycle [󰀳]. Te reducedascorbate is used as an exclusive coactor 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 apowerul 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 multiunctional enzymes glutathione
 S
-transerases (GSs)[󰀱󰀲], establishing the now widely accepted role o GSs as cellhousekeepers involved in the detoxi󿬁cation o endogenousas well as exogenous substances. More recently, several GSisoenzymes have been shown to modulate cell signalingpathways that control animal cell prolieration and cell death(apoptosis) [󰀵, 󰀱󰀳]. Blockage o the GSH/GS detoxi󿬁cation 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 alsoinvolvedinremovaloorganicperoxidesandH
2
O
2
 usingGSHorthioredoxinsaselectrondonors[󰀳].TeGSH/(GSH+GSSG) ratio, considered as GSH redox state, is likely to be armore in󿬂uential in the antioxidant deense, controlling geneexpressionsandthionylationprocessthantheabsolutesizeo the glutathione pool [󰀳].Cysteine (Cys), oneo the vitalbuilding blocks o GSH, isthe 󿬁rst stable and committed molecule in plant metabolismthat contains both sulur and nitrogen, synthesized by theO-acetylserine (thiol)lyase (OAS-L) either in ree activehomodimer or in association with serine acetyltranserase(SA)asaninactivesubunitothecysteinesynthasecomplex[󰀱, 󰀱󰀴]. OAS-L is the most enigmatic enzymatic systems in sulur metabolisms and its isoorms 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 signi󿬁cance in maintain-ingCyshomeostasisandthisisundertightregulationsunderstressul environments [󰀱󰀶󰀱󰀸]. Studies indicate that Cys degradation is mainly mediated through the desulurationo L-Cys and D-Cys by L-cysteine desulydrase (LCD) andD-cysteine desulydrase (DCD), respectively, rather thanpromoting Cys biosynthesis in high plants [󰀱󰀸]. Te resultanthydrogen sul󿬁de 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 prolierationwith 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 prolieration are two importantphenomena in plant developmental process [󰀲󰀲󰀲󰀴]; while learolling is supposed to be an adaptation to droughttolerance mechanisms, prolierative growth in stems, buds,and in other plant organs ofen leads to asciation throughdisturbances in SAM. Although moderate lea rolling andasciationinplant partshave longbeen utilizedorcropyieldenhancement [󰀲󰀲], they are still regarded as an enigmaticprocess as nothing is known regarding intrinsic cellular andbiochemical processes involved in cell prolierations duringrolling and asciation. Most o the studies in shoot meristemprolieration 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-redoxstatusinregulationoplantgrowthevents,scantwork has been done in this direction [󰀲󰀵]. Also, no reportsare available regarding cytogenetic behavior o prolierative 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 researchhasbeenmaniestedthroughdevelopmentorobustcytogenetic 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 aswellasaxillarybudasciation,inM
2
 progenyoethylmethanesulonate- (EMS-) mutagenized population. Te mutantwas advanced to next generation through sel-pollinationto perorm 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 mitotic󿬁delity 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.Aferascertaininguniormityoseedageandhomozy-gosity,reshseedspresoakedinwateror󰀵hweretreatedwithreshly prepared 󰀰.󰀱󰀵%, 󰀰.󰀲󰀵%, and 󰀰.󰀵% aqueous solutiono ethylmethane sulonate (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
). Seled 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 wereollowed to grow healthy plant progeny. Phenotypes o about󰀲󰀰󰀰󰀰 M
2
 individuals were screened during winter o 󰀲󰀰󰀱󰀲and 󰀲󰀰󰀱󰀳. One plant with characteristic in-rolled lea󿬂ets andprolierationinstemandaxillarybudshasbeenisolatedinM
2
progeny o EMS- (󰀰.󰀲󰀵%, 󰀶h) treated population. Seeds (󰀴󰀲seeds)othisvariantplantwerecareullyharvestedandsowninthenextseasontoraiseM
3
 progeny.Temutantphenotypewastruebreeding,andbasedonmorphologicalpeculiarities,the mutant was designated as
 rlfL-󰀱
 (
Rolled lea󿬂et and fasci-ated Lathyrus mutant-󰀱
). Further biochemical and molecularcharacterizations o the mutant were perormed on theseprogeny plants.
󰀲.󰀲. Culture Conditions and BSO reatment Protocol.
 Grasspea seeds obtained rom M
3
 plants o 
 rlfL-󰀱
 mutant and itsmother variety BioL-󰀲󰀱󰀲 were surace-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 suloximine, 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 speci󿬁c or each enzyme under chilledconditions. Te homogenate was squeezed through ourlayers o cheese cloth and centriuged 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 perormed 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 sul󿬁de (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 thereleaseosul󿬁deromCysinatotalvolumeo󰀱mLconsistingo 󰀲.󰀵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󰀱󰀰󰀰
󽠵
Lo󰀳󰀰mMFeCl
3
dissolved in 󰀱.󰀲NHCl and 󰀱󰀰󰀰
󽠵
L o 󰀲󰀰mM N,N-dimethyl-p-phenylenediamine dihydrochloride dissolved in 󰀷.󰀲NHCl[󰀳󰀵]. Te ormation o methylene blue was determined at󰀶󰀷󰀰nm.SolutionswithdifferentconcentrationsoNa
2
Swerepreparedandtreatedinthesamewayastheassaysamplesandwereusedorthequanti󿬁cationoenzymaticallyormedH
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 EDA, 󰀱mM D, and 󰀲% (w/v) polyvinylpyrrolidone using a chilled mortar and pestle kept in an icebath. Te homogenate was centriuged at 󰀱󰀵,󰀰󰀰󰀰g at 󰀴
C or󰀲󰀰min. Clear supernatant was used or enzyme assays. Formeasuring APX activity, the tissue was separately ground in

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