γ -aminobutyric acid transaminase deficiency impairs central carbon metabolism and leads to cell...

g-Aminobutyric acid transaminase deficiency impairs centralcarbon metabolism and leads to cell wall defects during saltstress in Arabidopsis roots

HUGUES RENAULT1,2*, ABDELHAK EL AMRANI2, ADELINE BERGER3, GRÉGORY MOUILLE3,LUDIVINE SOUBIGOU-TACONNAT4, ALAIN BOUCHEREAU1 & CAROLE DELEU1

1IGEPP, UMR1349 INRA, Université de Rennes 1, F-35653 Le Rheu, France, 2ECOBIO, UMR6553 CNRS, Université deRennes 1, F-35042 Rennes, France, 3Institut Jean-Pierre Bourgin, UMR1318 INRA, AgroParisTech, F-78026 Versailles, Franceand 4URGV, UMR INRA1165, Université d’Evry Val d’Essonne, ERL CNRS 8196, F-91057 Evry, France

ABSTRACT

Environmental constraints challenge cell homeostasis andthus require a tight regulation of metabolic activity. We havepreviously reported that the g-aminobutyric acid (GABA)metabolism is crucial for Arabidopsis salt tolerance asrevealed by the NaCl hypersensitivity of the GABAtransaminase (GABA-T, At3g22200) gaba-t/pop2-1 mutant.In this study, we demonstrate that GABA-T deficiencyduring salt stress causes root and hypocotyl developmentaldefects and alterations of cell wall composition. A compara-tive genome-wide transcriptional analysis revealed thatexpression levels of genes involved in carbon metabolism,particularly sucrose and starch catabolism, were found toincrease upon the loss of GABA-T function under salt stressconditions. Consistent with the altered mutant cell wall com-position, a number of cell wall-related genes were also founddifferentially expressed. A targeted quantitative analysis ofprimary metabolites revealed that glutamate (GABA precur-sor) accumulated while succinate (the final product ofGABA metabolism) significantly decreased in mutant rootsafter 1 d of NaCl treatment. Furthermore, sugar concentra-tion was twofold reduced in gaba-t/pop2-1 mutant rootscompared with wild type. Together, our results provide strongevidence that GABA metabolism is a major route for succi-nate production in roots and identify GABA as a majorplayer of central carbon adjustment during salt stress.

Key-words: Arabidopsis thaliana; GABA; glutamate;polyamines; salt tolerance; succinate; sugars; TCA cycle.

INTRODUCTION

Salt stress increasingly threatens agriculture and food pro-duction worldwide. However, the molecular determinantsand mechanisms underlying salt tolerance are still not wellunderstood. Salt accumulation in soils decreases water avail-ability and hampers plant nutrition leading to secondary

stresses (e.g. oxidative stress). In consequence, plants sufferfrom photosynthesis inhibition, disturbed metabolic homeos-tasis and cell structure damages, which reduce plant growthand fertility. Arabidopsis thaliana is considered sensitive tosalt stress (Munns & Tester 2008). In response to salt, itadopts a strategy aimed at alleviating impact of stress oncellular activity. This is achieved by controlling several fun-damental processes such as ion transport, metabolic activity,cell wall remodelling and cell fate determination.

g-Aminobutyric acid (GABA) is a four-carbon, non-protein amino acid found in almost all living organisms. Themolecule has been intensively investigated in mammals inwhich it acts as a neurotransmitter in the central nervoussystem (Owens & Kriegstein, 2002). In plants, it is well estab-lished that GABA accumulates in response to a wide rangeof environmental cues, including salt stress (Shelp et al., 1999;Kinnersley & Turano, 2000). GABA metabolism takes placein two different cellular compartments. GABA synthesisoccurs in the cytosol from glutamate as a result of glutamatedecarboxylase activity (GAD, Supporting InformationFig. S1; Turano & Fang 1998). Subsequently, after transport,GABA is degraded in two steps in the mitochondrion. Thefirst step of GABA degradation involves GABA transami-nase (GABA-T, Supporting Information Fig. S1) that cataly-ses transamination of GABA using pyruvate or glyoxylate asamino group acceptor (Clark et al. 2009). The GABA-Tencoding gene (At3g22200) is present as a single copy in theArabidopsis genome and was initially termed pollen-pistilincompatibility 2 (POP2; Palanivelu et al. 2003). The secondstep of GABA degradation leads to the production of succi-nate by the succinic semialdehyde dehydrogenase (SSADH,Supporting Information Fig. S1; Busch & Fromm 1999).

Features of GABA metabolism are consistent with aninvolvement in anaplerosis that can restore the tricarboxylicacid (TCA) cycle succinate pool (Supporting InformationFig. S1). Recent advances in the understanding of the meta-bolic function of GABA support such a function. Forinstance, it was reported, in tomato leaves and potato tubers,that GABA metabolism can compensate inhibition of theTCA cycle, providing an alternative anaplerotic route(Studart-Guimarães et al. 2007; Araújo et al. 2008). Moreo-ver, GABA metabolism was shown to be responsible for asignificant proportion of succinate synthesis for the TCA

Correspondence: H. Renault. Fax: +33 3 68 85 19 21; e-mail: [email protected]

*Current address: Institut de Biologie Moléculaire des Plantes,CNRS UPR2357, Université de Strasbourg, 12 rue du GénéralZimmer, F-67084 Strasbourg, France.

Plant, Cell and Environment (2013) 36, 1009–1018 doi: 10.1111/pce.12033

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cycle in illuminated leaves of Xanthium strumarium (Tch-erkez et al. 2009), and was furthermore proposed to be asubstrate for respiration during tomato fruit ripening (Yinet al. 2010).

We have previously published the physiological and meta-bolic characterization of the gaba-t/pop2-1 mutant upon saltstress (Renault et al. 2010). Based on root growth assays, weshowed that the mutant is oversensitive to the ionic compo-nent of salt stress. Besides, we demonstrated that, aftermidterm salt treatment (i.e. 4 d), gaba-t/pop2-1 roots accumu-late amino acids and are depleted in sugars. In the presentstudy, we aimed at (1) refining the phenotypic analysis of themutant after midterm salt treatment; and (2) investigatingtranscriptional and metabolic changes in the mutant in earlystep of salt stress (i.e. 1 d) in order to uncover determinants ofgaba-t/pop2-1 sensitivity to salt. We demonstrate here thatpop2-1 root and hypocotyl developments are impaired inresponse to salt stress and display alterations of the cell wallcomposition. A comparative microarray analysis reveals dif-ferential expression of cell wall- and carbon metabolism-related genes in the mutant compared with wild type (WT) insaline conditions. Metabolic profiling indicates that mutantroots contain less sugars and succinate upon salt treatment,and accumulate higher levels of glutamate compared withWT.

MATERIALS AND METHODS

Plant material and growth conditions

A. thaliana Landsberg erecta ecotype (Ler) was used as WT.Seeds of the gaba-t/pop2-1 mutant (Palanivelu et al. 2003)were provided by the Nottingham Arabidopsis Stock Centre(NASC, UK). Unless otherwise stated, plants were grownin vitro in square plates using a modified Hoaglandmedium (half-strength macronutrients) solidified with agaras reported before (Renault et al. 2010). Growth chamberparameters were set as follows:12 h/12 h day/night cycle (lightintensity of 100 mmol m-2 s-1), 22 °C and 60% relative humid-ity. For hydroponic culture, seeds were sown on 0.65% agar(w/v), stratified for 2–3 d, and moved to hydroponics 1 weeklater. Growth conditions, including salt treatment, were thesame as for in vitro cultures; nutrient solutions were renewedweekly and thoroughly aerated.

Scanning electron microscopy

Root tips were dissected using a razor blade and fixed for48 h at 4 °C in 2.5% glutaraldehyde solution prepared with0.1 m potassium phosphate buffer (pH 7.2). Next, sampleswere dehydrated with a graded ethanol series, dried withliquid CO2 using the critical point method and mounted onblack tape. Sample metallization was performed by sputter-ing gold palladium using a JEOL JFC 1100 device (JEOLSAS, Croissy-sur-Seine, France). Observations were carriedout with a JEOL JSM 6301F scanning electron microscope(SEM). Mutant and WT hypocotyls were directly imagedusing a HITACHI TM-1000 SEM (Hitachi GmbH, Krefeld,Germany).

Fourier-transform infrared microspectroscopy

Fourier-transform infrared (FT-IR) analyses were carried outat the ‘Plateau Technique de Chimie du Végétal’ (OC028INRA; Institut Jean-Pierre Bourgin, UMR1318 INRA/AgroParisTech) as described earlier (Renault et al. 2011). Inbrief, for each genotype/treatment, four Petri plates wereprepared and were randomly distributed within the growthchamber. Five seedlings per plate were then randomlysampled for the analysis (i.e. total of 20 seedlings). Seedlingswere squashed between two BaF2 slides and an area corre-sponding to the side of the hypocotyl’s central cylinder wasselected for FT-IR microspectroscopy. Spectra were collectedby a Thermo-Nicolet Nexus spectrometer (Thermo Scientific,Rockford, IL, USA) equipped with a Continuum microscopeaccessory (Thermo Scientific). Baseline correction and nor-malization of spectra were carried out as described inMouille et al. (2003).

JIM5 immunolocalization

Root tips were hand cut using razor blades.Sections were fixedin 4% (w/v) p-formaldehyde for 1 h in 50 mm PIPES buffer,pH 6.9, 5 mm MgSO4, and 5 mm ethylene glycol tetraaceticacid (EGTA) (microtubule stabilizing buffer (MTSB)] for30 min, then in 6.5 mm Na2HPO4, 1.5 mm KH2PO4, pH 7.3,14 mm NaCl, and KCl [phosphate buffered saline (PBS)] for15 min. Tissues were dehydrated in a graded ethanol series[30, 50, 70, 90 and 97% (v/v) in PBS]. Samples were incubatedin a mixture of 100% wax and 97% ethanol (1:1, v/v) at 40 °Covernight, and embedded in 100% wax at 40 °C for 2 h. Sec-tions (8 mm) were prepared using a microtome and air driedon polylysine-coated glass slides. Samples on slides weredewaxed and rehydrated through a degraded ethanol series[97, 90 and 50% (v/v) in PBS]. Samples were blocked with 1%(w/v) bovine serum albumin in PBS (blocking solution), andincubated with rat antibody against JIM5 epitope (Knox et al.1990; Willats et al. 2000) diluted in blocking solution. Afterthree washes of 5 min each in PBS, slides were incubated withgoat anti-rat IgG labelled with Alexa 488 (Molecular Probes,Eugene, OR, USA) in blocking solution. After washing withPBS, slides were sealed. As controls, sections were incubatedwith pre-immune sera as the primary antibodies or withoutthe primary antibodies, and with Alexa 488-labelled second-ary antibodies. Immunofluorescence was observed using aspectral confocal laser-scanning microscope (Leica TCS SP2AOBS).

Microarray analysis

The microarray data presented here are a part of a projectpreviously validated and partially published (Renaultet al. 2011). Data are publicly available at CATdb (http://urgv.evry.inra.fr/CATdb/; Project: AU05-03_GABA) andGene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/; GEO Accession No.: GSE11129). Procedures followedin the present study are the same as reported previously(Renault et al. 2011). Briefly, RNA was isolated from

1010 H. Renault et al.

© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 36, 1009–1018

10-day-old Ler and gaba-t/pop2-1 seedlings grown understandard conditions (control) and from 11-day-old seedlingstreated for 24 h with 150 mm NaCl (NaCl) using the SV totalRNA isolation Kit (Promega, Madison, WI, USA). For eachgenotype/treatment, two plates were prepared (i.e. two bio-logical replicates) and were randomly distributed within thegrowth chamber. Each sample consisted of approximately 30seedlings derived from one plate. For each biological repli-cate, one technical replicate with fluorochrome reversal wasperformed (i.e. a total of 4 hybridizations per comparison).Genes were considered as being differentially expressedbetween Ler and gaba-t/pop2-1 when Bonferroni-adjustedP-value was below 0.05.

qRT-PCR analysis

For each genotype/treatment, three plates were prepared (i.e.three biological replicates) and randomly distributed withinthe growth chamber. Shoots and roots of approximately 30seedlings derived from one plate were then harvested sepa-rately and ground using a mixer mill (MM 400;Retsch GmbH,Haan, Germany). Total RNA was isolated from 30 mg of theresulting plant material using the SV Total RNA Isolation kit(Promega) and subsequently treated with DNase I with theTURBO DNA-free kit (Applied Biosystems, Foster City, CA,USA). About 200 ng of total RNA was reverse transcribedusing an oligodT and the Taqman® Reverse TranscriptionReagents kit (Applied Biosystems). Quantitative PCR reac-tions were carried out as described previously (Renault et al.2010).Fold differences in expression were calculated using theDDCt method and the PP2AA3 gene as internal reference.Allprimers are listed in Supporting Information Table S1.

Metabolite analysis

WT and gaba-t/pop2-1 plants were grown together hydro-ponically. For each treatment, three identical but independ-ent hydroponic systems were used (i.e. three biologicalreplicates) and randomly distributed within the growthchamber. Plant samples, consisting of eight plants derivedfrom one hydroponic system, were harvested, snap frozen inliquid nitrogen, and freeze dried before homogenization witha mixer mill (MM 400; Retsch). Metabolite extraction wasperformed using a methanol:chloroform:water protocolaccording to Renault et al. (2010).Amino acids were analysedon a UPLC-PDA system as described previously (Renaultet al. 2010). Quantitative determination of sugars, sugar alco-hols and organic acids was carried out using a GC-FID device(Lugan et al. 2009). Metabolite levels are expressed in micro-moles per gram of plant dry weight (mmol·g-1 DW).

RESULTS

Root and dark-grown hypocotyl development isaltered in the gaba-t/pop2-1 mutant duringsalt stress

We previously reported that root growth of the GABA-Tdeficient gaba-t/pop2-1 mutant is severely affected by NaCl

treatment compared with WT plants (Renault et al. 2010). Inaddition, salt stress was found to strongly enhance POP2gene expression in root tips (Renault et al. 2010). The firstaim of this work was to further investigate root phenotypesof gaba-t/pop2-1 seedlings grown for 4 d on NaCl. While nodifference with WT was observed under standard growthconditions, gaba-t/pop2-1 seedlings displayed root swellingand twisting phenotypes under high salt (Fig. 1a). Uponcloser examination via scanning electron microscopy (SEM)mutant roots appeared thicker compared with WT. This rootswelling phenotype was confined to tissues above the divi-sion area (Fig. 1c) where POP2 was previously found to behighly expressed (Renault et al. 2010). Furthermore, SEMrevealed on gaba-t/pop2-1 roots, salt-induced wall degrada-tion and detachments of epidermal cells, likely as a result ofcell adhesion defects (Fig. 1c). SEM images of roots grownunder control conditions did not reveal any differencebetween WT and gaba-t/pop2-1 (Supporting InformationFig. S2).

Given the root growth inhibition in gaba-t/pop2-1 uponsalt (Renault et al. 2010) and the cell elongation inhibitoryeffect of GABA itself (Renault et al. 2011), we nextaddressed the question whether NaCl could differentiallyimpact cell elongation in the mutant. Dark-grown hypocotylswere selected as a model because their growth only reflectsthe cell elongation process (Gendreau et al. 1997). Moreover,gaba-t/pop2 was shown to be expressed in this organ(Renault et al. 2011). Hypocotyl growth assays revealed thatthe gaba-t/pop2-1 mutant was slightly, yet significantly (Stu-dent’s t-test, P-value < 0.001) hypersensitive to 50 mm NaClcompared with WT (Fig. 1d,e), suggesting a weak defect incell elongation. However, at NaCl concentrations exceeding50 mm, the length of hypocotyl of WT and gaba-t/pop2-1seedlings was undistinguishable (Fig. 1d,e). Despite thisweak effect of salt on mutant hypocotyl growth, a phenotypereminiscent of the root developmental defects describedabove could be observed on gaba-t/pop2-1 hypocotyls. Insupport, a SEM approach revealed that hypocotyls of etio-lated mutant displayed cell wall defects on 50 and 100 mmNaCl (Fig. 1e).

Hypocotyl and root cell wall compositions arealtered in gaba-t/pop2-1 during salt stress

Dark-grown hypocotyls are suitable for FT-IR microspec-troscopy cell wall fingerprinting (Mouille et al. 2003). FT-IRanalysis was therefore carried out on hypocotyl of etiolatedseedlings grown in the presence of 50 mm NaCl for com-parison of WT and mutant spectra. This showed that NaCltriggered significant changes in cell wall composition of themutant, especially between 1640 and 1770 cm-1 (Fig. 2a,b).These wavenumbers might stand for vibrations of esterbounds of esterified pectin and carboxyl bounds of non-esterified pectins (Sene et al. 1994). We next tested whetherpectins were affected in roots of the mutant during saltstress. To this end, the JIM5 epitope, specific to partiallymethyl-esterified homogalacturonan residues of pectins(Knox et al. 1990; Willats et al. 2000), was marked by

GABA and central metabolism during salt stress 1011


immunolocalization in salt-treated root tips using specificantibody and indirect immunofluorescence. Upon salt treat-ment, the JIM5 epitope was found to be less abundant inmutant compared with WT roots (Fig. 2c,d), confirminga decrease in partially methyl-esterified pectin levels ingaba-t/pop2-1 roots.

Differential response to salt stress ofpolyamines-, carbon metabolism- and cellwall-related genes in gaba-t/pop2-1

To identify the molecular determinants of gaba-t/pop2-1NaCl sensitivity, we compared transcription profiles of WTand gaba-t/pop2-1 seedlings grown under standard and saltstress conditions (i.e. 24 h, 150 mm NaCl). Total RNA wasisolated from whole seedlings and two biological replicateswere analysed by CATMA microarray (Crowe et al. 2003;Hilson et al. 2004). For details about experimental design andprocedures, see Materials and Methods section and Support-ing Information Fig. S3. Under standard growth conditions,Ler and gaba-t/pop2-1 transcriptomes were almost identical,indeed, solely nine genes were found differentially expressed(i.e. Bonferroni-adjusted P-value < 0.05; Supporting Infor-mation Appendix S1). Upon 24 h NaCl treatment, however,the expression of 172 genes was found different in the mutantcompared with WT (Supporting Information Appendix S1 &Fig. S3). The gene set, differentially expressed under saltstress conditions without being affected under standardgrowth conditions (168 out of 172 loci), was subjected to afunctional classification using the Classification SuperViewer

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Figure 1. Root and dark-grown hypocotyl development is altered in gaba-t/pop2-1 during salt stress. (a) Phenotype of 14-day-old seedlingstreated for 4 d with, or without (control), 150 mm NaCl. (b, c) SEM images (¥50 magnification) and close-ups (¥250 magnification) of wildtype (WT) (b) and pop2-1 (c) root extremities after salt stress (scale bar, 10 mm). (d) Phenotype of 4-day-old seedlings grown in dark onmedia supplemented with 0, 50 or 100 mm NaCl (scale bar, 5 mm). (e) SEM images (¥500 magnification) of etiolated WT and pop2-1hypocotyls (scale bar, 100 mm). Values below SEM images are means � SE of relative hypocotyl lengths (% of control). For eachexperimental condition the length of over 100 hypocotyls was measured.

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Figure 2. Cell wall modifications in the gaba-t/pop2-1 mutantduring salt stress. (a, b) Fourier-transform infrared (FT-IR) analysisof pop2-1 cell wall (Mouille et al. 2003) on 4-day-old hypocotylsgrown in the dark on agar media supplemented, or not (control),with 50 mm NaCl. Under each condition, FT-IR spectra wereacquired from 19 to 21 hypocotyls derived from four independentbiological replicates. (a) Average spectra. (b) Student’s t-test:t-value for the comparison between wild type (WT) and pop2-1(y-axis) is plotted against the wavenumbers (x-axis). Redhorizontal lines refer to the P = 0.95 significance threshold.Wavenumbers of chosen t-value peaks are indicated.(c, d) Immunolocalization of the homogalacturonan-specificJIM5 epitope in 14-day-old WT (c) and pop2-1 (d) roots after4 d of NaCl (150 mm) treatment (scale bar, 50 mm). Close-ups onsimilar WT and pop2-1 root zones are provided.



software in combination with MapMan terms (http://bar.utoronto.ca/ntools/cgi-bin/ntools_classification_superviewer.cgi). ‘Polyamine metabolism’ was uncovered as the mostenriched term (Table 1). Indeed, three genes encodingenzymes involved in polyamine biosynthesis (i.e.ARGININE DECARBOXYLASE 1, ACAULIS 5 andS-ADENOSYL-METHIONINE DECARBOXYLASE 1)

were found to be differentially expressed in the mutant uponhigh salinity (Table 2). The second most enriched term was‘Major CHO metabolism’ (Table 1). More specifically, theexpression of genes involved in sucrose and starch metabo-lism was found to be increased in gaba-t/pop2-1 upon saltstress (Table 2). Furthermore, the functional classificationrevealed a significant enrichment of the ‘Cell wall’ term(Table 1). Among these, three genes of cellulose biosynthesisdisplayed increased expression levels (i.e. CELLULOSESYNTHASE 1, CELLULOSE SYNTHASE 3, KORRI-GAN1) while the expression of two pectate lyases was foundto be decreased (Table 2). In addition, a number of genesencoding cell wall proteins were differentially regulated inthe mutant in response to high salinity (Table 2). A graphicaloverview of the transcriptional changes affecting loci impli-cated in central metabolism of gaba-t/pop2-1 is available inSupporting Information Fig. S4.

Next, the subcellular localization was assigned in silico toproteins encoded by differentially expressed genes using theonline tool Aramemnon (http://aramemnon.botanik.uni-koeln.de/; Supporting Information Fig. S5). A set of randomgenes was used as control. Strikingly, over 60% of the pro-teins encoded by down-regulated loci in gaba-t/pop2-1 inresponse to salt stress were related to the secretory pathway,while only 17% are attributed to secretion among a randomprotein encoding gene population (Supporting InformationFig. S5). These findings are consistent with the cell wall

Table 1. Enriched MapMan terms in the microarray dataset

MapMan term Frequencya Elementsb P-value

Polyamine metabolism 37.4 3 6.50E-05Major CHO metabolism 7.82 4 1.60E-03Metal handling 6.72 3 9.18E-03Cell wall 3.72 10 3.09E-04Hormone metabolism 2.61 7 0.013Transport 2.48 13 1.59E-03

The functional classification of genes was performed using the Clas-sification SuperViewer software (http://bar.utoronto.ca/ntools/cgi-bin/ntools_classification_superviewer.cgi) with MapMan terms.Input consisted of the 168 genes differentially expressed in the gaba-t/pop2-1 mutant during salt stress (i.e. transcriptionally sable incontrol conditions). Only the six most enriched terms with significantP-values (P-value < 0.05) are listed.aFrequency is normalized to overall ID number within Arabidopsisgenome.bNumber of genes matching the term.

Table 2. Differentially expressed polyamine-, carbon metabolism- and cell wall-related genes in gaba-t/pop2-1 upon salt stress

Locus Annotation Involved in pop2-1/Ler ratioa P-valueb

Polyamines metabolismAt2g16500 ADC1 Polyamine biosynthesis +0.86 5.55E-6At3g02470 SAMDC1 Polyamine biosynthesis +0.82 4.42E-5At5g19530 ACAULIS5 Polyamine biosynthesis -0.80 8.88E-5

Carbon metabolismAt4g02280 SUS3 Sucrose degradation +0.89 1.53E-6At5g48300 ADG1 Starch synthesis +0.70 4.89E-3At5g18670 BMY3 Starch degradation +0.83 1.82E-5At3g46970 PHS2 Starch degradation +0.78 2.20E-4At1g06410 TPS7 Trehalose synthesis +0.89 1.59E-6At1g42970 GAPB Glyceraldehyde-3-phosphate metabolism +0.74 1.37E-3At3g60750 Transketolase Pentose-phosphate metabolism +1.26 0.00E+0At4g15530 PPDK Pyruvate metabolism +0.78 2.50E-4

Cell wallAt5g49720 KOR1 Cellulose biosynthesis +0.77 3.72E-4At4g32410 CESA1 Cellulose biosynthesis +0.95 5.91E-8At5g05170 CESA3 Cellulose biosynthesis +0.69 8.03E-3At1g04680 Pectate lyase Cell wall degradation -0.97 1.55E-8At4g24780 Pectate lyase Cell wall degradation -1.30 0.00E+0At4g28250 EXPB3 Cell wall loosening -0.76 4.39E-4At2g03090 EXPA15 Cell wall loosening -0.65 3.59E-2At3g55500 EXPA16 Cell wall loosening -0.92 2.84E-7At4g01630 EXPA17 Cell wall loosening +0.90 6.43E-7At4g40090 AGP3 Cell wall protein -0.76 4.18E-4

Genes significantly more or less expressed in the mutant compared with wild type (WT) are indicated by positive and negative pop2-1/Ler ratios,respectively.aExpressed in log2.bBonferroni-adjusted P-value.



alteration observed in the mutant upon salt stress as cell wallelaboration critically relies on secretion of proteins andbuilding blocks.

Sucrose degradation is transcriptionallyup-regulated in gaba-t/pop2-1 roots uponsalt stress

Microarray analysis identified a number of genes differen-tially expressed in response to salt stress in gaba-t/pop2-1whole seedlings. To analyse if the altered gene expressioncan be specifically associated to molecular imbalancesoccurring in root tissues, where gaba-t/pop2 is predomi-nantly expressed (Renault et al. 2010) and its loss of func-tion has the greatest impact (Fig 1a,c), the transcriptionanalysis was refined on RNA isolated from shoots androots, respectively. The expression of three genes found dif-ferentially expressed in response to high salinity in gaba-t/pop2-1 according to the microarray data was followed.Thus, CYCLIN-DEPENDENT KINASE B2; 1 (CDKB2;1;cell division), SUCROSE SYNTHASE 3 (SUS3; sucrosedegradation) and BETA-AMYLASE 3 (BMY3; starchdegradation) were targeted for qRT-PCR analysis. In allinstances, the most substantial transcriptional differencesbetween WT and gaba-t/pop2-1 were recorded in roots(Fig. 3). For instance, CDKB2;1 expression was found to betwofold reduced in gaba-t/pop2-1 roots after salt treatmentcompared with control conditions, but did not respond tosalt stress in WT roots (Fig. 3a). Thus, as a consequence ofhigh salinity cell division might be slowed down in mutantroots. Similarly, SUS3 gene expression in response to saltwas unaffected in WT but strongly enhanced by salt treat-ment in mutant roots (Fig. 3b). Finally, BMY3 was foundtranscriptionally up-regulated upon salt exposure in WTand gaba-t/pop2-1 roots; however, the induction was signifi-cantly stronger in mutant roots (Fig. 3c).

Glutamate accumulates while sugars andsuccinate levels decrease in gaba-t/pop2-1 rootsduring salt stress

In response to salt treatment, a drastic reduction in sugarconcentrations has been reported in gaba-t/pop2-1 mutantroots (Renault et al. 2010). In our previous study, the meta-bolic profiling was carried upon 4 d of salt treatment. Here, todetermine early metabolic changes caused by salt stress, wequantified sugars, organic and amino acids in roots and shootsof WT, and gaba-t/pop2-1 upon 24 h of salt treatment (i.e.150 mm NaCl).As a convenient way to get sufficient amount ofroot material for metabolic analyses, we used 30-day-oldhydroponically grown plants.At first sight,only sugars showeda contrasted response to salt stress in gaba-t/pop2-1, both inshoots and in roots (Fig. 4a). They were slightly more abun-dant in gaba-t/pop2-1 shoots than in WT after salt treatment.Conversely, sugar concentration was almost reduced by a halfin mutant roots compared with WT after salt stress (Fig. 4a),which is in agreement with our previous metabolic analysisperformed after 4 days of stress on 14-day-old seedlings(Renault et al. 2010). No significant change in amino acids andorganic acids in overall levels could be observed (Fig. 4b,c).Acloser look to individual metabolite levels confirmed thatsucrose concentration was almost twofold reduced in mutantroots after salt stress (Table 3). Interestingly, it also pointed tocontrasted responses of glutamate and succinate to salt stressin mutant roots. Glutamate accumulated in mutant rootsunder saline conditions, rising by 40% compared with WT(Table 3), while succinate decreased by a similar proportion(Table 3). Several other metabolites showed significantchanges in their abundances.For instance,glutamine level wastwo times lower in gaba-t/pop2-1 roots after salt stress, sug-gesting an impairment of nitrogen assimilation or recycling inthe mutant. The next question was whether the overallnitrogen/carbon balance was affected in the mutant duringsalt stress. However, element analysis did not reveal any sig-nificant change in composition of the mutant after 4 d of salttreatment (Supporting Information Table S2).

DISCUSSION

The first clear response of plants to salt stress is a reducedgrowth and photosynthetic activity as salt accumulates inleaves and rises to toxic levels (Munns 2002). Meanwhile,cells undergo an extensive metabolic reprogramming toadapt to saline conditions and reach new homeostasis(Hasegawa et al. 2000; Lugan et al. 2010; Krasensky & Jonak2012). In a context of reduced photosynthetic activity, alter-native energy metabolisms become of primary importance.Several studies indicate that salt stress enhances respiration,a response that might contribute to increase salt tolerance(Jacoby, Taylor & Millar 2011; Millar et al. 2011). As a conse-quence of impaired carbon fixation, respiration uses a set ofalternative substrates (i.e. lipids and proteins) as energysupply (Araújo et al. 2011). In heterotrophic organs, such asroots and etiolated hypocotyls, the respiration process is evenof greater importance.

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Figure 3. Differential response in gene expression to salt stress ingaba-t/pop2-1 roots. Expression levels of CYCLIN-DEPENDENTKINASE B2; 1 (CDKB2; 1; a), SUCROSE SYNTHASE 3 (SUS3;b) and BETA-AMYLASE 3 (BMY3; c) were analysed byqRT-PCR in shoots and roots of 11-day-old seedlings treated, ornot, for 24 h with 150 mm NaCl. Values are mean � SE folddifferences between saline and control conditions (untreatedseedlings). For each condition, means were calculated from threeindependent biological replicates. Statistical significance wascalculated by Student’s t-test: *P < 0.05; ***P < 0.001.



A previous study demonstrated that salt stress enhancesGABA metabolism (Renault et al. 2010) suggesting itsinvolvement in salt response. In addition, a prominent func-tion was attributed to the GABA metabolism in Arabidopsisroots (Renault et al. 2010). GABA metabolism can contrib-ute to the activity of both the TCA cycle and the respiratoryelectron transfer chain by generating succinate and NADHthrough SSADH activity (Busch & Fromm 1999; SupportingInformation Fig. S1). Thus, it can constitute a gateway forprotein respiration, or at least for glutamate utilization uponphotosynthesis inhibition. Recently, a mitochondrial GABApermease has been characterized and shown to bridgeGABA metabolism and TCA cycle (Michaeli et al. 2011).This work demonstrates that proper GABA transport intothe mitochondrion, where its degradation occurs (SupportingInformation Fig. S1), is required for plant growth uponcarbon limitation (Michaeli et al. 2011). These findingssuggest a role for GABA in the respiration under low sugarconditions. Here we show that suppression of GABA-T activ-ity resulted in a significant decrease of succinate in roots after24 h salt treatment (Fig. 4 and Table 3). Simultaneously,glutamate levels were found significantly increased, indicat-ing that GABA metabolism was a major route for succinateproduction from glutamate. However, we cannot rule outthat these responses were secondary effects of GABA-T defi-ciency as 24 h was our earliest time point. An anapleroticfunction was also attributed to glutamate dehydrogenaseduring dark-induced carbon starvation (Miyashita & Good2008), especially in roots (Fontaine et al. 2012). It thusappears that Arabidopsis adapts to carbon limitation byinducing alternative anaplerotic pathways, with glutamate asa major player. This is consistent with the recently proposednon-cyclic nature of TCA cycle (Tcherkez et al. 2009). Inaddition, it was reported that GABA production is rapidly

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CitrateMalateFumarateSuccinate

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WT

pop2

-1W

T

pop2

-1W

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pop2

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pop2

-1

Control NaCl Control NaCl

Shoots Roots

Figure 4. Targeted and quantitative metabolic analysis ofgaba-t/pop2-1. Quantitative determination of sugars (a), organicacids (b) and amino acids (c) in shoots and roots of 30-day-oldhydroponically grown wild type (WT) and pop2-1 plants. Prior toharvesting, plants were treated 24 h with 150 mm NaCl. Results aremeans � SE of three independent biological replicates. ‘Others’include all protein amino acids except glutamate (Glu), glutamine(Gln) and proline (Pro).

Table 3. Comparison of metabolite levels in gaba-t/pop2-1 andWT roots after salt stress

Metabolite level (mmol g-1 DW)

Control NaCl

WT pop2-1 WT pop2-1

Sucrose 35.9 48.7 130.5 *70.1Glucose 42.8 25.8 68.4 27.4Fructose 21.8 8.9 26.5 17.2Citrate 44.3 40.2 92.3 62.1Succinate 7.8 8.1 8.4 *5.2Malate 77.6 81.9 77.1 *104.7GABA 6.7 *15.2 6.9 *28.4Glutamate 23.9 23.3 44.4 *60.8Glutamine 14.8 *13.5 44.7 *24.5myo-Inositol 4.9 5.2 5.1 *3.8

Absolute values of metabolite levels in WT and pop2-1 roots after24 h of 150 mm NaCl treatment. Indicated values are means of threeindependent biological replicates; standard errors are illustrated inFig. 4. Asterisks indicate significant differences between mutant andWT (t-test, P < 0.05) under given conditions.DW, dry weight; GABA, g-aminobutyric acid; WT, wild type.



activated under stress through post-translational regulationof GAD activity by Ca2+/calmodulin complex (Baum et al.1993; Ling et al. 1994; Snedden et al. 1995). GABA metabo-lism thus seems to be a source of succinate mobilized underfluctuating and stressful conditions to help buffering TCAcycle activity.

Intriguingly, in the gaba-t/pop2-1 mutant, we observedup-regulation of genes involved in sucrose and starch deg-radation upon salt stress (Table 2, Fig. 3). Transcriptionalchanges are expected to precede alterations in metaboliteconcentrations, but they may also reflect feedback regula-tion of transcription by metabolic status. It is thus possiblethat gaba-t/pop2-1 plants compensated for the defect inGABA metabolism by remobilizing carbon reserves tofeed the glycolytic pathway and maintain the TCA cycle(Fig. 5). The severely reduced sugar levels recorded in gaba-t/pop2-1 roots upon 24 h and 4 d salt treatment (Fig. 4 and

Table 3; Renault et al. 2010) might be the trade-off forhomeostasis. Moreover, sugar starvation markers (DARK-INDUCIBLE 1 and DARK-INDUCIBLE 6; SupportingInformation Appendix S1) were found overexpressed in themutant suggesting that the amount of carbohydrates con-sumed during the stress period was critical for plant sur-vival. Interestingly, the mitochondrial GABA permeasemutant gapb exhibits an enrichment in nitrogen-rich com-pounds and a depletion in carbon-rich molecules, especiallysugars, under low light (Michaeli et al. 2011). Here, thedescribed results, in agreement with previously publisheddata, suggest that under low carbon conditions a disturbedGABA metabolism results in reduced sugar concentrations.

Furthermore, the gaba-t/pop2-1 mutant displayed cell walldefects upon salt stress (Figs 1 & 2). The impairment of celladhesion observed in gaba-t/pop2-1 might be a consequenceof the reduced pectin level scored in mutant roots (Fig. 1c;Bouton et al. 2002). A recent study illustrates the deleteriousimpact of TCA cycle impairment on cell wall elaboration intomato (van der Merwe et al. 2010).The authors propose thatunder energy-limited conditions, plants prioritize metabolicpathways that support energy production. As a consequence,metabolic flux can be diverted from cell wall productionresulting in cell wall defects and subsequently in disruptedroot growth (van der Merwe et al. 2010). These findings areconsistent with ours because cell wall alterations were onlyobserved in the root zones elongating upon salt stress, sug-gesting that the availability of cell wall precursors was thelimiting factor preventing root growth (Fig. 5). In support ofthis hypothesis, we observed significantly reduced levels ofthe pectin precursor myo-inositol (Loewus & Murthy 2000)in mutant roots after salt stress (Table 3). A defect in proteinglycosylation has previously been shown to induce salt sen-sitivity as well as cell wall alterations (Koiwa et al. 2003; Kanget al. 2008; Zhang et al. 2009). We thus tested if proteinglycosylation was affected in the gaba-t/pop2-1 mutant.However, we did not detect any changes in the electro-phoretic mobility of KORRIGAN1 (Supporting InformationFig. S6), an N-glycosylated protein (Nicol et al. 1998; Kanget al. 2008) encoded by a differentially expressed locus in thegaba-t/pop2-1 mutant (Table 2).

We observed a differential transcriptional regulation ofgenes involved in polyamine metabolism in response to saltstress in gaba-t/pop2-1.The GABA production via putrescineoxidation has been a matter of debate for decades (Flores &Filner 1985) and remains questioned (Shelp et al. 2012).Recently, diamine oxidases have been proposed to participatein GABA accumulation during salt stress in soybean roots(Xing et al. 2007). Considering the reported regulatory role ofpolyamines (Bouchereau et al. 1999), two hypotheses can beproposed to explain the differential response of polyaminegene transcription in gaba-t/pop2-1. Firstly, a metabolic feed-back regulation could be induced in consequence of alteredGABA degradation. The second possibility would be adevelopmental feedback regulation in response to thegrowth defects observed in gaba-t/pop2-1. For instance, thesalt-induced transcriptional down-regulation of ACAULIS5, encoding a thermospermine synthase involved in cell

TCA

cycle

GABA*

Citrate*

Isocitrate

-Ketoglutarate

Succinyl-CoA

Succinate*

Fumarate*

Malate*

Oxaloacetate

Pyruvate

Sucrose* Starch

Glutamate*

SSA

Glucose*

Fructose*

x

Glutamine* Amino acids*

Proteolysis

Cell wallprecursors

myo-Inositol*

Nitrogen assimilation

Figure 5. Relationships between GABA and central metabolismsin gaba-t/pop2-1 roots during salt stress. The model was inferredfrom data obtained in our previous work (Renault et al. 2010) andthe present study. Reduced metabolite levels or metabolicstep/pathway activities in pop2-1 compared with wild type (WT)are marked in blue, while enhanced levels or activities areindicated in red. Metabolite levels that were experimentallyassessed, either previously (Renault et al. 2010) or in this study, areindicated by asterisks. Dashed arrows indicate multistep reactions.



elongation and vasculature formation, might be the result ofsuch a feedback adjustment (Clay & Nelson 2005; Muñiz et al.2008).

In conclusion, our study further confirmed the functionalinvolvement of the GABA metabolism in salt stress toler-ance. Moreover, we provided evidence for the implication ofGABA in central carbon metabolism regulation in rootsunder stress conditions. This function appeared to be criticalas GABA-T deficiency resulted in dramatic root cell walldegradation upon salt stress.The obtained results open excit-ing perspectives for further investigations of metabolic con-tribution of GABA to the central metabolism. Futureprospects could benefit from utilization of the gaba-t/pop2-1mutant combined with flux analyses.

ACKNOWLEDGMENTS

The authors are very grateful to Joseph Le Lannic (Univer-sity of Rennes 1), Sylvie Citerne (INRA, Versailles), HalimaMorin (INRA, Versailles) and Martine Gonneau (INRA,Versailles) for their help with scanning electron microscopy,FT-IR analysis, cell wall epitope immunolocalization andKOR1 Western blot, respectively. We gratefully acknowledgeDrs Danièle Werck-Reichhart, François Bernier and AnneMolitor for the critical reading of the manuscript. H.R. wassupported by the ‘Ministère de l’Enseignement Supérieur etde la Recherche’.

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Received 25 September 2012; received in revised form 31 October2012; accepted for publication 31 October 2012

SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article:

Figure S1. Schematic representation of GABA metabolism.Figure S2. SEM images of WT and gaba-t/pop2-1 roots understandard growth conditions.Figure S3. Overview of microarray experiment.Figure S4. Graphical representation of genes identified bymicroarray analysis and related to central metabolism.Figure S5. Subcellular localization of proteins encoded bygenes identified by microarray.Figure S6. KORRIGAN1 Western blot analysis.Table S1. qRT-PCR primer pairs used in the study.Table S2. Element analysis of gaba-t/pop2-1 roots and shootsafter salt stress.Appendix S1. List of differentially expressed genes in gaba-t/pop2-1 mutant.



γ -aminobutyric acid transaminase deficiency impairs central carbon metabolism and leads to cell...

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