methodology article open access an improved, low-cost

Alatorre-Cobos et al BMC Plant Biology 2014 1469httpwwwbiomedcentralcom1471-22291469

METHODOLOGY ARTICLE Open Access

An improved low-cost hydroponic system forgrowing Arabidopsis and other plant speciesunder aseptic conditionsFulgencio Alatorre-Cobos12 Carlos Calderoacuten-Vaacutezquez13 Enrique Ibarra-Laclette14 Lenin Yong-Villalobos1Claudia-Anahiacute Peacuterez-Torres1 Araceli Oropeza-Aburto1 Alfonso Meacutendez-Bravo14 Sandra-Isabel Gonzaacutelez-Morales1Dolores Gutieacuterrez-Alaniacutes1 Alejandra Chacoacuten-Loacutepez15 Betsy-Anaid Pentildea-Ocantildea1 and Luis Herrera-Estrella1

Abstract

Background Hydroponics is a plant growth system that provides a more precise control of growth mediacomposition Several hydroponic systems have been reported for Arabidopsis and other model plants The ease ofsystem set up cost of the growth system and flexibility to characterize and harvest plant material are featurescontinually improved in new hydroponic system reported

Results We developed a hydroponic culture system for Arabidopsis and other model plants This low cost proficientand novel system is based on recyclable and sterilizable plastic containers which are readily available from localsuppliers Our system allows a large-scale manipulation of seedlings It adapts to different growing treatments and hasan extended growth window until adult plants are established The novel seed-holder also facilitates the transfer andharvest of seedlings Here we report the use of our hydroponic system to analyze transcriptomic responses ofArabidopsis to nutriment availability and plantpathogen interactions

Conclusions The efficiency and functionality of our proposed hydroponic system is demonstrated in nutrient deficiencyand pathogenesis experiments Hydroponically grown Arabidopsis seedlings under long-time inorganic phosphate (Pi)deficiency showed typical changes in root architecture and high expression of marker genes involved in signaling andPi recycling Genome-wide transcriptional analysis of gene expression of Arabidopsis roots depleted of Pi by shorttime periods indicates that genes related to general stress are up-regulated before those specific to Pi signaling andmetabolism Our hydroponic system also proved useful for conducting pathogenesis essays revealing early transcriptionalactivation of pathogenesis-related genes

Keywords Hydroponics Arabidopsis Root Phosphate starvation Pathogenesis

BackgroundStandardization of growth conditions is an essential fac-tor to obtain high reproducibility and significance in ex-perimental plant biology While lighting humidity andtemperature are factors that can be effectively controlledby using plant growth chambers or rooms media compos-ition can be significantly altered by the physiochemicalcharacteristics and elemental contaminants of differentbatches of gelling agents [12]

Correspondence lherreralangebiocinvestavmx1Laboratorio Nacional de Genoacutemica para la Biodiversidad (Langebio)Unidadde Genoacutemica Avanzada (UGA) Centro de Investigacioacuten y Estudios Avanzadosdel IPN 36500 Irapuato Guanajuato MeacutexicoFull list of author information is available at the end of the article

copy 2014 Alatorre-Cobos et al licensee BioMedCreative Commons Attribution License (httpdistribution and reproduction in any mediumDomain Dedication waiver (httpcreativecomarticle unless otherwise stated

For example the inventory of changes in root systemarchitecture (RSA) as a plant adaptation to nutrient stresscan be influenced by the presence of traces of nutrients indifferent brands or even batches of agar as reported for thePi starvation response [1] Detailed protocols for obtainingreal nutrient-deficient solid media for several macro andmicronutrients have been recently reported [12] Theseprotocols describe a careful selection of gelling agentsbased on a previous chemical characterization that increasethe cost and time to set up experiments In addition thoseproblems associated with media composition plant growthwindow is reduced in petri plates (maximum 2ndash3 weeks)[3] In vitro culture time can be extended using glass jars

Central Ltd This is an Open Access article distributed under the terms of thecreativecommonsorglicensesby20) which permits unrestricted use provided the original work is properly credited The Creative Commons Publicmonsorgpublicdomainzero10) applies to the data made available in this

Figure 1 Hydroponic system component dimensions andassembly A and B) Dimensions and assembly of seed-holder C)Assembled hydroponic system Containers with different volume forliquid media are shown The numbers at the bottomrsquos containerindicate the maximum volume and the number inside the containerthe volume of liquid media used in each case

Alatorre-Cobos et al BMC Plant Biology 2014 1469 Page 2 of 13httpwwwbiomedcentralcom1471-22291469

but accessibility to the root system is then compro-mised Furthermore additional handling and thus unneces-sary plant stress during seedlings transfer to new growthmedia as well as during plant material collection shouldbe also considered when experiments on solid media aredesignedOne strategy for circumventing all problems described

above is the use of hydroponic systems for plant cul-ture Several hydroponic systems have been reported forArabidopsis [4-13] and some of them are now commer-cially available (Aeroponicsreg) [12] Most of these systemsare integrated by a plastic glass or polycarbonate containerwith a seed-holder constituted by rock wool a polyureth-ane (sponge) piece a steel or nylon mesh polyethylenegranulate or a polyvinyl chloride (PVC) piece Those areopen systems which allow axenic conditions or reducedalgal contamination into liquid growth media but sterilityis not possibleHere we describe step by step a protocol for setting up a

simple and low-cost hydroponic system that allows steril-ity conditions for growing Arabidopsis and other modelplants This new system is ideal for large-scale manipula-tion of seedlings and even for fully developed plants Oursystem is an improved version of Schlesier et al [8] inwhich the original glass jar and steel seed-holder are sub-stituted by a translucent polypropylene (PE) container anda piece of high-density polyethylene (HDPE) mesh Allcomponents are autoclavable reusable cheap and readilyavailable from local suppliers The new device designed asseed-holder avoids the use of low-melting agarose as sup-port for seeds allowing a quick and easy transfer to newmedia conditions andor harvest of plant material The ef-ficiency and functionality of our proposed system is dem-onstrated and exemplified in experiments that showedtypical early transcriptional changes under Pi starvationand pathogen infection

Results and discussionDescription of the hydroponic systemWe have improved a previously reported hydroponicsystem consisting of a glass jar and stainless piece inte-grated by a wire screen fixed between two flat rings andheld in place by three legs [8] by a simpler and cheapersystem assembled with a PE vessel and a seed-holder in-tegrated by a circle-shape HDPE mesh and two PE rings(Figure 1AB Table 1) Vessels and mesh used here arereadily available in local markets vessels are actuallyfood containers (Microgourmetreg Solo Cup USA wwwsolocupcom) available in food package stores while theHDPE mesh is a piece of anti-aphid mesh acquired inlocal stores providing greenhouse supplies (wwwtextilesagricolasmx) A small cotton plug-filled orifice in thecontainer lid allows gas exchange to the system (Figure 1C)This ventilation filter reduces but does not eliminate high

humidity in the medium container Such problem could besolved adding more ventilation filters or using other sealingmaterials as micropore 3 Mreg paper tape Aeration of the li-quid medium is not required for our hydroponic systemNo negative effects on plant growth have been observedwhen small tanks are used as medium containers (refer-ences in Table 1)The new seed-holder for positioning seeds on top of

the liquid medium consists of a mesh of HDPE monofil-aments held between two PE rings (ring A and B) withan area of 7854 cm2 (diameter =10 cm) which is able tohold 50 to 65 Arabidopsis seedlings for up to 10ndash15 daysafter germination (Figure 1AB Figure 2) (Table 1) Fullydeveloped Arabidopsis plants (2ndash3 plants per vessel) canalso be grown in this system if the container lid is replacedby another PE container (Additional file 1) Anti-aphidmesh with a 075 mm by 075 mm opening size (mesh usu-ally named 25 times 25) is adequate for keeping Arabidopsisseeds on top of the mesh (Figure 2AB) and allowing inde-pendent root system development (Figure 2CDE) Anti-aphid or anti-insect mesh with lower density can be usefulfor seeds larger than Arabidopsis seeds No legs for sup-porting the mesh-holder are needed in our hydroponicsystem The seed-holder is just placed into the container

Table 1 Comparison between hydroponic systems previously reported and the system proposed here

Parameter Agar-filledplastic holder

Rockwool-filledplastic holder

Sponge into apolypropylene sheet

Polyethylene granulate Stainless mesh fixed twometal rigsNylon mesh on

photo slide mount

This system

Liquid medium container Plastic box Plastic box Magenta GA-7 vesselreg Glass vessel Round-rim glass jarsglass vessel Plastic container

Costs Intermediate to high Intermediate High High High Low

Setup time Intermediate to high Intermediate Low Low High Low to intermediate

Reuse of seed-holder No No No No YesNo Yes

Throughput Intermediate Intermediate High High Highintermediate Intermediate

Container volume Small to high Small to intermediate Small Small to high Intermediate Intermediate to high

Medium evaporation High High Low High LowHigh Low

Seedling number per holder One One One Many Many Many

Sterility No No Yes No YesNo Yes

Aeration YesNo YesNo No No No No

Time for moving and sampling largebatches of plants between media

High High High High High Low

Development window Adult plants Adult plants Seedling to adult plants Seedling Seedling Seedling to adult plants

References [3912] [41011] [6] [7] [5813]

Alatorre-C

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Biology20141469

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Figure 2 Arabidopsis seedlings growing under the hydroponic system proposed A) Seeds sown on the meshrsquos seed-holder A close-upview of a single seed is shown (inset) B-E) Seedlings growing in our hydroponic system Top (B) and lateral view (C) of 12-day-old seedlings Top(D) and lateral view (E) of 3-weeks-old seedlings


and kept in place by pressing against the container wallsUnlike other protocols previously reported (Table 1) thecontainer size of the system described here can vary ac-cording to volume of medium required (Figure 1C)However the same standard seed-holder can be used for1000 ml 750 ml or 500 ml containers giving an effectivevolume for root growth of 600 ml 350 ml and 210 ml re-spectively (Figure 1C)Our hydroponic system can be used for growing other

model species under aseptic conditions Solanum lyco-persicum Nicotiana tabacum and Setaria viridis seedswere sterilized and directly sowed on the mesh For allspecies an adequate growth of shoot and root systemwas observed two weeks after germination (Figure 3)

Figure 3 The hydroponic system proposed can be used with other mshoot growth of S lycopersicum N tabacum and S viridis at 2 to 3 weeks o

Other advantages of this hydroponic system are relatedto plant transfer and plant tissue collection For bothonly a dressing tissue forceps (6 or 12 inch) previouslysterilized is required to pull up the seed-holder and placeit into new media (Figure 4A) or to submerge it into a li-quid nitrogen container for tissue harvest (Figure 4B) Rootharvest of young seedlings of the hydroponic system is alsoeasier and less time-consuming than those from seedlingsgrown in agar media When the seed-holder is taken outfrom the container young roots adhere to mesh and canbe blotted with an absorbent paper towel and immediatelyfrozen in liquid nitrogen Shoot biomass can be also easilydetached from the mesh using a scalpel and then the meshwith the attached roots can be processed separately

odel monocot and dicot plants Lateral and top views of root andld

Figure 4 An easy and quick transfer to new growth media andor root harvesting can be carried out with this hydroponicssystem A) Tobacco seedlings are transferred handling the seed-holderonly B) Batch of tobacco seedlings growing on the seed-holder frozeninto liquid nitrogen


Protocol for setting up the hydroponic systemStep by step instructions for set up of hydroponic systemare indicated in the following section and the Additionalfile 2 Tips and important notes are also indicated

1 Getting a nylon mesh (See Figure 1A)

Get a piece of anti-aphid or anti-insect mesh Drawa circle (10 cm diameter) using a marker and acardboard template Trim the circle using a forkAfter tripping the circle remove color traces onmesh using absolute alcohol Wash the mesh underrunning water (Option Use deionized water) Dryon paper towels Tip Use a red color marker fordrawing Red color is easier to clean than other colors

2 Making a mesh holder (See Figure 1AB)

Cut the 500 ml PE containers bottom Use ascalpel blade Leave a small edge (05 cm width)The mesh circle will put on this edge For ring Aleave a height of 25 cm for ring B leave 3 cm TipUse a scalpel blade with straight tip to cut easilythe containers bottom

3 Preparing the container lid

Locate the center of container lid and mark it Drillthe lid center Seal the small lid hole with a cottonplug Tip Use a hot nail to melt a hole in the lid toavoid burrs

4 Sterilization

Container and rings and mesh have to be separatelysterilized by autoclaving (121degC and 15 psi pressureby 20 minutes) Put container ring and meshgroups into poly-bags For container and ringsclose but not seal the poly-bags If so pressurevariations during sterilization could damage themImportant point Put the autoclave in liquid media

mode Tip After sterilization put poly-bags intoanother bag for reducing contamination risks

5 Hydroponic system assembly (See Figure 1C)

Open the sterilized poly-bags containing containersrings meshes and lids Put a volume of previouslysterilized liquid medium into the container Tip theuse liquid media at room temperature reduces thesteam condensate on container lid and walls Take aring B with a dressing tissue forceps and put it intothe container just above the liquid media level Puta mesh piece on the ring B lift it slowly and thenreturn it on the ring avoiding to form bubbles Fitthe ring A onto the mesh piece Tip If it is difficultto fit the ring A onto the mesh piece warm thering quickly using a Bunsen burner Finally closethe container

Applications of our hydroponic system 1) Quicktranscriptional responses to Pi starvationApplications of this new hydroponic culture system formodel plants were analyzed in this study Changes duringPi starvation at the transcriptional level associated withthe Arabidopsis RSA modifications have been previouslydescribed [13] Here first we compared the effects of Pi-availability on RSA and the expression profiles of eightmarker genes for Pi deficiency in Arabidopsis seedlingsgrown in hydroponics versus agar media Then taking ad-vantage of the short time that is required with this newhydroponic system for transferring plants to differentmedia early transcriptional responses to Pi depletion wereexplored at the genome-wide level such responses havenot been previously evaluated

Arabidopsis growth and Pi-depletion responsive genes onPi-starved hydroponic mediaArabidopsis seeds were germinated and grown for 12 daysin hydroponics or agar media containing high-Pi (125 mM)or low-Pi (10 μM) concentrations as previously re-ported [1415] By day 12 after germination a higher shootand root biomass was produced by Arabidopsis seedlingsgrown in hydroponics than those grown in solid media(Figure 5AB) which is consistent with previous com-parisons between both methods for growing Arabidopsis[5] The typical increase in root biomass accumulationunder Pi stress was observed in seedlings grown in agarmedium however such change was not statistically signifi-cant (Figure 5B) In contrast the dry weight of roots ofseedlings grown in hydroponics under Pi stress was 225-fold higher compared to that observed for Pi-sufficientseedlings (Figure 5B) This higher root growth under low-Pi is a typical RSA change that allows an increase of Pi up-take under natural soil conditions [14] Regarding RSAadaptation to low Pi availability we also found a 30 re-duction in primary root length with respect to control

Figure 6 qRT-PCR expression profiling of marker genes for Pistarvation in Arabidopsis seedlings grown hydroponicallyExpression profiling of A) transcriptional modulators and genesinvolved with root meristem growth and B) Pi signaling and recyclingArabidopsis seedlings were grown for 21 days under two different Piregimens (minusP = 10 μM Pi +P = 125 mM Pi) RNA of whole seedlings wasextracted at six time points and gene expression levels were analyzed byqRT-PCR assays Relative quantification number (RQ) was obtained fromthe equation (1 + E)2ΔΔCT where ΔΔCT represents ΔCT(minusP)ndashΔCT(+P) andE is the PCR efficiency CT value was previously normalized using theexpression levels of ACT2 PPR and UBHECT as internal reference Datapresented are means plusmn SE of three biological replicates (n = 100-150)

Figure 5 Plant growth under hydroponics or solid media undercontrasting Pi regimens (A-F) Arabidopsis seedlings were directlysowed on the seed-holder (50 ndash 60 seed per mesh) or agar media(30ndash35 seeds per plate) growth for 12 days under two different Piregimens (minusP = 10 μM Pi +P = 125 mM Pi) and then analyzed Barsrepresent means plusmn SE (Hydroponics biological replicates = 5 n = 20ndash60agar medium biological replicates = 10ndash15 n = 15) Asterisks denote asignificant difference from corresponding control (+P treatment)according Studentrsquos t test (Plt005)


treatment under hydroponics while such reduction washigher (76) in roots from agar media (Figure 5C) Simi-larly there was a modest increase in lateral root and roothair density under low-Pi in liquid media whereas amarked increase under same Pi growth condition wasfound in agar media (Figure 5DEF)Although the effects of Pi deficiency on root develop-

ment were more severe in agar media than in our hydro-ponic system the typical root modifications induced byPi stress (primary root shortening and higher productionof lateral roots and root hairs) [14] were observed inboth systems Differences in the magnitude of RSA alter-ations in response to Pi-deprivation could be explainedby variations in medium composition caused by gellingagents added andor the ease to access to Pi available inthe growth systems used It has been previously shownthan contaminants such as Pi iron and potassium in thegelling compounds can alter the morphophysiological andmolecular response to Pi starvation [1] Hydroponics pro-vides a better control on media composition and allows adirect and homogenous contact of the whole root systemwith the liquid medium This condition could be improve

nutrient uptake and under Pi starvation alleviate the dra-matic changes of RSA observed usually in roots of seed-lings grown in agar mediaAfterwards we determined the efficiency of the hydro-

ponics system for inducing expression of low-Pi-responsivegenes Analysis of the expression profiles for eight genesinvolved with transcriptional metabolic and morphologicalresponses to Pi starvation were carried out in whole Arabi-dopsis seedlings that were grown in either low or high-Pihydroponic conditions at 4 7 12 14 17 and 21 days Tran-script level quantification of the transcriptional factors(TF) PHR1 (PHOSPHATE STARVATION RESPONSE 1)WRKY75 (WRKY family TF) and bHLH32 (basic helix-loop-helix domain-containing TF) revealed a direct influ-ence of Pi stress persistence on the up-regulation of thesethree molecular modulators [16-18] WRKY75 had thehighest expression level among the TFs analyzed with asignificant induction in expression after 12 days under Pideficiency (Figure 6A) BHLH32 showed a similar increasein expression As most molecular responses to Pi starvationare affected in phr1 mutant PHR1 has been considered a


master controller of Pi signaling pathway [1619] In thecase of PHR1 we found that this gene did not show as con-stitutive expression under Pi deficiency as originally re-ported [16] Instead the expression profile of this masterregulator in roots showed responsiveness to low-Pi condi-tions (Figure 6A) These data are consistent with the lowtranscriptional induction of PHR1 previously observed inArabidopsis shoots [20] LPR1 (LOW PHOSPHATE 1) andPDR2 (PHOSPHATE DEFICIENCY RESPONSE 2) twogenes involved in root meristem growth [21] and the E2ubiquitin conjugase PHOSPHATE 2 (PHO2UBC24) re-lated with Pi loading [22] showed a notable increase in ex-pression after 14 d of treatment (Figure 6A) In contrastSPX1 (a gene encoding a protein with a SYG1Pho81XPHR1 domain) and PLDZ2 (PHOSPHOLIPASE DZ2) twotypical marker genes of Pi deficiency implicated with Pi sig-naling and recycling [2324] respectively showed a signifi-cant induction starting at day four Both SPX1 and PLDZ2but especially SPX1 had a marked increase in expressionlevel (Figure 6B) The expression analysis of these Pi-responsive genes together with RSA analyses during Pi star-vation on hydroponics demonstrate the high performanceof our system for plant growing and for analyzing molecu-lar responses to nutrimental deficiency

Exploring early genome-wide transcriptional responses toPi depletion overview and functional classification ofdifferentially expressed genesEarly transcriptional responses to Pi availability at thegenome-wide level (4 h to lt12 h) have been previouslydetermined in whole Arabidopsis seedlings using micro-array platforms [2526] An important experimental con-dition in those studies has been the use of a 100ndash200μM as a low-Pi concentration considered enough tosupport biomass accumulation but not to induce an ex-cessive Pi accumulation [26] It has been reported thatArabidopsis seedlings growing at 100 μM Pi in agarmedia had similar endogenous phosphorus (P) biomassproduction and RSA to those growing at 1 mM Pi [14]In liquid media 200 μM Pi has also been considered asa Pi-sufficient condition for growing monocot speciessuch as maize [27] We found that Arabidopsis seedlingsgrown with150 μM Pi in liquid media are not able to in-duce the expression of AtPT2AtPHT14 (PHOSPHATETRANSPORTER 2) a high-affinity Pi transporter respon-sive to Pi starvation reviewed in [28] as revealed by ana-lysis of Arabidopsis seedlings harboring the transcriptionalAtPT2GUS reporter Seedlings growing in hydroponicsduring 12 days showed null expression of the reporter ineither shoot or root When these seedlings were trans-ferred to Pi-depleted media AtPT2GUS reporter was de-tected 12 h after transfer (Figure 7)In order to demonstrate the efficiency of our system

to elucidate early transcriptional responses Arabidopsis

seedlings were germinated and grown in the hydropon-ics system with 125 μM Pi during 12 days and then im-mediately deprived of Pi Samples were taken at threeshort-time points (10 min 30 min and 2 h) (Figure 8A)Roots were harvested and frozen immediately after eachtime point total RNA extracted and their transcriptomeanalyzed by microarray expression profiling For data ana-lyses differences in gene expression between Pi-depletedversus Pi-sufficient roots were identified (the overall Pavailability effect) and also the differences caused by the Piavailability by time interaction (time times Pi effect) Accord-ing to the stringency levels used (FDRle 005 and fold plusmn2)a total of 181 genes showed differential expression in atleast one of three sampled time points (see Additionalfile 3) A total of 92 genes were found to be up-regulatedand 89 down-regulated by Pi-depletion (Figure 8B) Inter-estingly only 3 genes out of the 92 induced and 1 down-regulated out of the 89 repressed were common to allthree time points evaluated thus indicating specific tran-scriptional responses depending of the time point analyzed(Figure 8B) When clustered into functional classifica-tions (Table 2 and Additional file 3) some resembledthose previously reported [25-2729] thus validating oursystem for high throughput transcriptional analysesAccording to the expression profile up-regulated geneswere clustered in six different groups whereas only threegroups were identified for repressed genes (Additionalfile 3) Analysis of expression patterns by agglomerativehierarchical clustering showed a high number of up-regulated genes in the last time point evaluated (2 h) whilean opposite tendency was observed for down-regulatedgenes which were more responsive in the first time point(10 min) (Figure 8C) Differentially expressed genes wereclassified into functional categories according to TheMunich Information Center for Protein Sequences classifi-cation (MIPS) using the FunCat database [30] Categoriesmore represented in up-regulated genes were those relatedwith Metabolism Transcription Protein metabolism andInteraction with the environment (Table 2) Also therewas a similar number of induced and repressed genes inPi phospholipid and phospholipid metabolism categor-ies with the exception of those related with glycolipidmetabolism Interestingly the Energy category (glycolysisgluconeogenesis pentose-phosphate pathway respirationenergy conversion and regeneration and light absorption)was only represented in induced genes (Table 2)

Early transcriptional responses to low Pi availabilityinvolves cell wall modifications protein activityoxidation-reduction processes and hormones-mediatedsignaling that precede the reported Pi-signaling pathwaysAccording to the functional annotation of the ArabidopsisInformation Resource database (TAIR at wwwarabidopsisorg) most genes either induced or repressed during the

Figure 8 Early changes in the transcriptome of Arabidopsis roots under Pi starvation A) Workflow for experiments Arabidopsis seedlingswere grown hydroponically for 12 days under sufficient Pi level (125 μM Pi) and then transferred to Pi-depleted liquid media for short times Rootswere harvested RNA isolated and transcriptome analyzed using an oligonucleotide microarray platform B) Edwards-Venn diagrams showing commonor distinct regulated genes over the sampled time points C) Clustering of differentially expressed genes Clustering was performed using the Smoothcorrelation and average linkage clustering in GeneSpring GX 731 software (Agilent Technologies) Orange indicates up-regulated green indicatesdown-regulated and white unchanged values as shown on the color scale at the right side of the figure

Figure 7 AtPT2GUS expression pattern under Pi depletion Arabidopsis AtPT2GUS seedlings were grown hydroponically for 12 days undersufficient Pi level (125 μM Pi) and then transferred to Pi-depleted liquid media or control media (125 μM Pi mock) GUS activity in leaves and root wasmonitored by histochemical analyses at different time points GUS expression in the mock condition is shown for the last time sampled (48 h)


Table 2 Distribution of functional categories of differentially expressed genes responding to Pi-deprivation under shorttime points in Arabidopsis roots

Functional category Up-Down-regulated genes ()

Time point sampled

10 min 30 min 2 h

Metabolism 967 159 214714 341241

Energy 322- 107- 243-

Cell cycle and DNA processing 322227 357714 487689

Transcription 129454 714214 121103

Protein fate 3221354 714714 121137

Protein with binding function or cofactor requirement 161159 321714 219275

Regulation of metabolism and protein function - 357714 243344

Cell transport transport facilities and transport routes 967454 142714 731103

Cellular communicationsignal transduction mechanism -227 -142 -344

Cell rescue defense and virulence 322227 107142 975103

Interaction with the environment 967227 214214 243689

Systematic interaction with the environment 322227 357- 243689

Cell fate -227 - -

Development 645227 -142 -

Biogenesis of cellular components - 357714 243689

Subcellular localization 2934 357214 268344

Unclassified proteins 451431 214357 268413

Functional categories according to The Munich Information Center for Protein Sequences classification Differentially expressed genes with a fold change of atleast plusmn2 at any time point and FDR le005


first 30 min of Pi depletion are related to cell wall compos-ition protein activity oxidation-reduction and hormones-mediated signaling Previously known Pi-responsive genessuch MGDG SYNTHASE 3 (MGD3) SQDG SYNTHASE 2(SQD2) PURPLE ACID PHOSPHATASE 22 (PAP22) andS-ADENOSYLMETHIONINE SYNTHASE 1 (SAM1) pre-sented significant changes in expression until the last timepoint evaluated (2 h) Interestingly a few transcriptionalcontrollers were expressed differentially throughout the en-tire experimentAt 10 minutes Arabidopsis roots responded to Pi-

deprivation with the activation of 27 genes (185 of total)involved in polysaccharide degradation callose depositionpectin biosynthesis cell expansion and microtubule cyto-skeleton organization (see group I Additional file 3) Genesets related with oxidation-reduction processes proteinactivity modifications (ubiquitination myristoylation ATPor ion binding) and hormones-mediated signaling (absci-sic acid jasmonic acid) were also represented Overrepre-sentation of groups according functional processes wasnot clear in down-regulated genes excepting those relatedto modifications to protein fate (135 of total 44 genes)As Pi depletion progressed (30 min) transcriptional

changes related to cell wall decreased while responses toion transport signaling by hormones (auxins abscisicacid salicylic acid) or kinases were more represented in

both induced and repressed genes (Additional file 3) Indown-regulated genes this trend was also found in thelast time point (2 h) At 30 minutes interestingly genesinvolved with Pi-homeostasis eg SPX1 and GLYCEROL-3-PHOSPHATE PERMEASE 1 (G3Pp1) were already in-duced (see group IV and V Additional file 3)A higher number of up-regulated genes was found two

hours after Pi-depletion Most induced genes (9 out of37 genes) were related to ion transport or homeostasis butalso to carbohydrate metabolism oxidation-reduction sig-naling protein activity and development Importantly othertypical molecular markers for Pi starvation were also in-duced within 2 hours Two phosphatidate phosphatases(PAPs) (At3g52820 and At5g44020) were induced graduallyaccording Pi-starvation proceeded MGD3 and SQD2 bothinvolved with Pi recycling were also induced at 2 hours(see group VI Additional file 3) Expression of these genestogether with SPX1 and G3Pp1 indicate that the classicaltransduction pathways related with Pi-starvation can betriggered as early as two hours after seedlings are exposedto media lacking Pi SPX1 is strongly induced by Pi starva-tion and usually classified as member of a system signal-ing pathway depending of SIZ1PHR1 reviewed in [31] Itsearly induction (3ndash12 h) has been previously reported [25]however an ldquoimmediate-early responserdquo within few minutesafter Pi depletion has been not reported so far Likewise a

Alatorre-Cobos et al BMC Plant Biology 2014 1469 0 of 13httpwwwbiomedcentralcom1471-22291469

role for an enhanced expression of G3Pp1 inside trans-duction pathways or metabolic rearrangements trig-gered by Pi stress is still poorly understood [2526] Arecent functional characterization of Arabidopsis gly-cerophosphodiester phosphodiesterase (GDPD) familysuggests glycerol-3-phosphate (G3P) as source of Pi orphosphatidic acid (PA) which could be used by glycerol-3phosphatase (GPP) or DGDGSQDG pathways [32] Earlyinduced expressions of G3Pp1 PAP22 and MGD3 is inagreement with the hypothesis that under Pi deficiencyG3P could be first converted into PA by two acyltransfer-ase reactions and Pi would be then released during thesubsequent conversion of PA into diacylglycerol (DAG) byPAPs [32] DAG produced could be incorporated intoDGDG or SQDG by MGD23 and DGD12 and SQD12respectively [32] MGD2 and MGD3 have been found in-duced in Arabidopsis seedlings depleted of Pi for 3ndash12 h[25] This early transcriptional activity for MGD genesduring Pi starvation is also reflected in enhanced enzym-atic activities as revealed in Pi-starved bean roots [33] In-creased PA levels and MGDG and DGDG activities havebeen reported in bean roots starved of Pi for less than 4 h[31] Early gene expression activation of genes encodingMGDG and DGDG but not PLDC enzymes suggestsG3P and not PC as source for PA and DAG biosynthesisfor early Pi signaling and recycling pathwaysAccording with our data a specific transduction pathway

to Pi deficiency could be preceded by general responsesrelated to stress which could modify metabolism beforetriggering specific expression of transcriptional factorsThis idea is consistent with previous reports assayingPi-depletion in Arabidopsis by short and medium-longtimes (3ndash48 h) which also reported differentially expressedgenes related with pathogenesis hormone-mediated signal-ing protein activity redox processes ion transport andcell wall modifications [252834] Similar results have beenrecently reported in rice seedlings under Pi starvation for1 h [35]

Figure 9 PR1GUS expression pattern under P syringae pv tomato inand then transferred to liquid media containing P syringae bacteria (final 000monitored by histochemical analyses at different time points GUS expression

Applications of our hydroponic system 2) Pathologicalassays to evaluate systemic defense responsesIn order to determine the suitability of our hydroponicsystem to perform Arabidopsis-pathogen interactionswe evaluated the systemic effect of root inoculationwith Pseudomonas syringae pv tomato strain DC3000(Pst DC3000) on transcriptional activation of the pathogenesis-related gene PR1 Although P syringae is generallyknown as a leaf pathogen it has been proven to be an ex-cellent root colonizer in Arabidopsis [3637] TransgenicArabidopsis seedlings carrying the PR1GUS construct weregrown for 12 days and then inoculated with 0002 OD600 offresh bacterial inoculum β-glucoronidase (GUS) activitywas analyzed by histochemical staining at different timeintervals after inoculation Systemic response to Pst rootcolonization was evident between 2 and 6 hours afterinoculation (hai) as revealed by strong expression of themarker gene in leaves After 24 hai GUS activity spreadthroughout the whole shoot system but not in roots(Figure 9) These results demonstrate a good perform-ance for studying plant responses to pathogens

ConclusionsHere we describe a practical and inexpensive hydroponicsystem for growing Arabidopsis and other plants understerile conditions with an in vitro growth window that goesfrom seedlings to adult plants Our system uses recyclableand plastic materials sterilizable by conventional autoclav-ing that are easy to get at local markets In contrast to otherhydroponic systems previously reported the componentsof the system (container size mesh density lid) describedhere can be easily adapted to different experimental designsor plant species The seed-holder avoids the use of an agar-ose plug or any other accessory reducing time for settingup experiments and decreasing risks of contaminationApplications and advantages of our hydroponic system

are exemplified in this report First rapid transcriptomechanges of Arabidopsis roots induced by Pi depletion

cubation Arabidopsis PR1GUS seedlings were grown hydroponically2 OD600) or control media (mock) GUS activity in shoot and root wasin mock condition is shown for the last time point sampled (24 h)


were detected by a rapid harvest from growth mediausing our new seed-holder designed Our analyses con-firm that Arabidopsis roots early responses to Pi deple-tion includes the activation of signaling pathways relatedto general stress before to trigger those specific to Pistress and support the idea that G3P could be a sourceof Pi and other molecules as PA during early signalingevents induced by Pi starvation Second our hydroponicsystem showed a high performance to set up pathogen-esis assays

MethodsGrowth mediaFor solid or liquid media a 01 X Murashige and Skoog(MS) medium pH 57 supplemented with 05 sucrose(Sigma-Aldrich) and 35 mM MES (Sigma-Aldrich) wasused For solid growth media agar plant TC micropropaga-tion grade (compositionpurity not provided) (A296 Phyto-technology Laboratories US) from Gelidium species wasused at 1 (WV)

Plant material and growth conditionsArabidopsis thaliana ecotype Columbia (Col-0 ABRCstock No 6000 from SALK Institute) marker lines PR1GUS (At2g14610) (kindly provided by FM Ausubel)and AtPT2GUS (At2g38940) [38] tobacco cv Xhanti to-mato cv Micro-Tom and Setaria viridis seeds were used inthis study In all cases seeds were surface sterilized by se-quential treatments with absolute ethanol for 7 min 20(vv) commercial bleach for 7 min and rinsed three timeswith sterile distilled water Previous sterilization Setariaseeds were placed at minus80degC overnight as recommended[39] Arabidopsis sterilized seeds were vernalized at 4degC for2 days (solid media) or 4 days (hydroponics) For Arabidop-sis 50ndash65 seeds were directly sowed on the mesh of theseed-holder and 30ndash35 seeds in Petri plates (50 ml volume15 mm times 150 mm Phoenix Biomedical) Seedlings of allspecies evaluated were grown at 22degC except tobaccowhich was grown at 28degC using growth chambers (PercivalPerry IA USA) with fluorescent light (100 μmol mminus2 sminus1)and a photoperiod of 16 h light8 h dark

Analysis of root architecture traitsTo determine root architecture traits seedlings weregrown on agar plates at an angle of 65deg or under hydro-ponic conditions Root length was measured from root tipto hypocotyls base For lateral root (LR) quantification allclearly visible emerged secondary roots were taken intoaccount when the number of LRs was determined Roothair density was calculated from root images taken witha digital camera connected to an AFX-II-A stereomicro-scope (Nixon Tokyo) Statistical analysis of quantitativedata was performed using the statistical tools (Studentrsquos ttest) of Microsoft Excel software

GUS analysesFor histochemical analysis of GUS activity Arabidopsisseedlings were incubated for 4 h at 37degC in a GUS reac-tion buffer (05 mg mlminus1 of 5-bromo-4-chloro-3-indolyl-b-D-glucuronide in 100 mM sodium phosphate bufferpH 7) Seedlings were cleared using the method previ-ously described [40] At least 15 transgenic plants wereanalyzed and imaged using Normarski optics on a LeicaDMR microscope

Microarray analysisRoots were collected and immediately frozen and RNAisolated using the Trizol reagent (Invitrogen) and puri-fied with the RNeasy kit (Qiagen) following the manu-facturerrsquos instructions Spotten glass microarray slides(Arabidopsis Oligonucleotide Array version 30) were ob-tained from University of Arizona (httpagarizonaedumicroarray) Three biological replicates (150 seedlings perreplicate) were used for RNA isolation and two technicalreplicates (in swap) to the two channel microarrays Fluor-escent labeling of probes slide hybridization washing andimage processing was performed as described [27] A loopdesign was used in order to contrast the gene expressiondifferences between treatments and time points Micro-array normalization and data analysis to identify differen-tially expressed genes with at least two-fold change inexpression were carried as previously reported [27]The microarray data have been deposited in Gene Ex-

pression Omnibus (GEO) and are accessible through GEOSeries accession number GSE53114 (httpwwwncbinlmnihgovgeoqueryacccgiacc=GSE53114)

Transcript analysisTotal RNA was extracted with Trizol (Invitrogen) andpurified using Qiagen RNeasy columns according to themanufacturerrsquos instructions cDNA was synthesized using30 μg of total RNA with SuperScript III Reverse Transcript-ase (Invitrogen) and used for qRT-PCR (7500 Real TimePCR System Applied Biosystems) Expression of markergenes for Pi starvation was analyzed using the oligonucleo-tides listed in the Additional file 4 Gene expression ana-lyses were performed as previously reported [27] BrieflyRelative quantification number (RQ) was obtained fromthe equation (1 + E)2ΔΔCT where ΔΔCT represents ΔCT(Treatment)-ΔCT (Control) and E is the PCR efficiencyEach CT was previously normalized using the expres-sion levels of ACT2 (At3g18780) PPR (At5g55840) andUPL7 (At3g53090) as internal references

Pseudomonas syringae bioassaysBacterial strain P syringae pv tomato (Pst) DC3000 was cul-tured on Kingrsquos B medium (KB) supplied with 50 μg mlminus1

rifampicin PR1GUS seedlings were grown hydroponicallyas described above For infection of 12 day-old seedlings a


bacterial culture was grown overnight in KB at 28degC Bac-teria were centrifuged washed three times with sterilizedwater and resuspended in water to a final OD600 of 004The appropriate volume was added to seedling medium toa final OD600 of 0002 After infection plant material washarvested at several time points for GUS histochemical as-says and analyzed under a Leica DMR microscope

Additional files

Additional file 1 35-40-day-old Arabidopsis plants growing underour hydroponic system proposed The Arabidopsis seeds were directlysowed on the seed-holder and three adult plants per vessel were grownuntil the flowering began

Additional file 2 Video showing the assembly process for ourhydroponic system

Additional file 3 Differentially expressed genes by Pi depletionduring short time points in Arabidopsis roots

Additional file 4 qRT-PCR primers used in this study

AbbreviationsMGDG Monogalactosyldiacylglycerol DGDG DigalactosyldiacylglycerolSQDG Sulfoquinovosyldiacylglycerol

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsFAC CCV and LHE conceived the new hydroponic system and designed theexperiments FAC CCV EIL LYV C-APT AOA AMB S-IGM DAG and B-APOperformed the experiments FAC CCV EIL and LHE analyzed the data FACCCV and LHE drafted the manuscript All authors read and approved thefinal manuscript

AcknowledgementsThis work was partially funded by the Howard Medical Institute (Grant55003677) to LHE FAC is indebted to CONACYT for a PhD fellowship(190577) The authors would like to thank R Sawers (Langebio) for providingS viridens seeds J H Valenzuela for P syringae pv tomato strain J AntonioCisneros-Duraacuten (Cinvestav Unidad Irapuato) for his great help with photog-raphy and video in this work and Flor Zamudio-Hernaacutendez and Mariacutea de JOrtega-Estrada for microarray and qRT-PCR services We are grateful to theanonymous reviewers for their positive comments and meticulous revisionfor improving the quality of this manuscript

Author details1Laboratorio Nacional de Genoacutemica para la Biodiversidad (Langebio)Unidadde Genoacutemica Avanzada (UGA) Centro de Investigacioacuten y Estudios Avanzadosdel IPN 36500 Irapuato Guanajuato Meacutexico 2Current address Departmentof Biological and Environmental Sciences Institute of BiotechnologyUniversity of Helsinki 00014 Helsinki Finland 3Current address InstitutoPoliteacutecnico Nacional Centro Interdisciplinario de Investigacioacuten para elDesarrollo Integral Regional Unidad Sinaloa 81101 Guasave Sinaloa Meacutexico4Current address Red de Estudios Moleculares Avanzados Instituto deEcologiacutea AC Carretera Antigua a Coatepec 351 Xalapa 91070 VeracruzMeacutexico 5Current address Instituto Tecnoloacutegico de Tepic Laboratorio deInvestigacioacuten Integral en Alimentos Divisioacuten de Estudios de Posgrado 63175Tepic Nayarit Meacutexico

Received 15 December 2013 Accepted 13 March 2014Published 21 March 2014

References1 Jain A Poling MD Smith AP Nagarajan VK Lahner B Meagher RB

Raghothama KG Variations in the composition of gelling agents affect

morphophysiological and molecular responses to deficiencies ofphosphate and other nutrients Plant Physiol 2009 1501033ndash1049

2 Gruber BD Giehl RFH Friedel S von Wireacuten N Plasticity of the Arabidopsisroot system under nutrient deficiencies Plant Physiol 2013 163161ndash179

3 Conn SJ Hocking B Dayod M Xu B Athman A Henderson S Aukett LConn V Shearer MK Fuentes S Tyerman SD Gilliham M Protocoloptimising hydroponic growth systems for nutritional and physiologicalanalysis of Arabidopsis thaliana and other plants Plant Methods 2013 94doi1011861746-4811-9-4

4 Gibeaut DM Hulett J Cramer GR Seemann JR Maximal biomass ofArabidopsis thaliana using a simple low-maintenance hydroponic methodand favorable environmental conditions Plant Physiol 1997 115317ndash319

5 Toda T Koyama H Hara T A simple hydroponic culture method for thedevelopment of a highly viable root system in Arabidopsis thalianaBiosci Biotechnol Biochem 1999 63210ndash212

6 Arteca RN Arteca JM A novel method for growing Arabidopsis thalianaplants hydroponically Physiol Plant 2000 108188ndash193

7 Battke F Schramel P Ernst D A novel method for in vitro culture ofplants cultivation of barley in a floating hydroponic system Plant MolBiol Rep 2003 21405ndash409

8 Schlesier B Breacuteton F Mock H-P A hydroponic culture system for growingArabidopsis thaliana plantlets under sterile conditions Plant Mol Biol Rep2003 21449ndash456

9 Noreacuten H Svensson P Andersson B A convenient and versatile hydroponiccultivation system for Arabidopsis thaliana Physiol Plant 2004 121343ndash348

10 Robison MM Smid MPL Wolyn DJ High-quality and homogeneousArabidopsis thaliana plants from a simple and inexpensive method ofhydroponic cultivation Can J Bot 2006 841009ndash1012

11 Huttner D Bar-Zvi D An improved simple hydroponic method forgrowing Arabidopsis thaliana Plant Mol Biol Rep 2003 2159ndash63

12 Tocquin P Corbesier L Havelange A Pieltain A Kurtem E Bernier GPeacuterilleux C A novel high efficiency low maintenance hydroponic systemfor synchronous growth and flowering of Arabidopsis thalianaBMC Plant Biol 2003 32

13 Bargmann BOR Birnbaum KD Fluorescence activated cell sorting of plantprotoplasts J Vis Exp 2010 36 httpwwwjovecomdetailsphpid=1673doi1037911673

14 Loacutepez-Bucio J Hernaacutendez-Abreu E Saacutenchez-Calderoacuten L Nieto-Jacobo MFSimpson J Herrera-Estrella L Phosphate availability alters architecture andcauses changes in hormone sensitivity in the Arabidopsis root systemPlant Physiol 2002 129244ndash256

15 Peacuterez-Torres CA Loacutepez-Bucio J Cruz-Ramiacuterez A Ibarra-Laclette E Dharmasiri SStelle M Herrera-Estrella L Phosphate availability alters lateral root develop-ment in Arabidopsis by modulating auxin sensitivity via a mechanisminvolving the TIR1 auxin receptor Plant Cell 2008 123258ndash3272

16 Rubio V Linhares F Solano R Martiacuten AC Iglesias J Leyva A Paz-Ares JA conserved MYB transcription factor involved in phosphate starvationsignaling both in vascular plants and in unicellular algae Gene Dev 200152122ndash2133

17 Devaiah BN Karthikeyan AS Raghothama KG WRKY75 transcription factoris a modulator of phosphate acquisition and root development inArabidopsis Plant Physiol 2007 1431789ndash17801

18 Chen ZH Nimmo GA Jenkins GI Nimmo HG BHLH32 modulates severalbiochemical and morphological processes that respond to Pi starvationin Arabidopsis Biochem J 2007 405191ndash198

19 Bustos R Castrillo G Linhares F Puga MI Rubio V Peacuterez-Peacuterez J Solano RLeyva A Paz-Ares J A central regulatory system largely controls transcrip-tional activation and repression responses to phosphate starvation inArabidopsis PLoS Genet 2010 6e1001102

20 Nilsson L Muumlller R Nielsen TH Increased expression of the MYB-relatedtranscription factor PHR1 leads to enhanced phosphate uptake inArabidopsis thaliana Plant Cell Environ 2007 301499ndash1512

21 Ticconi CA Lucero RD Sakhonwasee S Adamson AW Creff A Nussaume LDesnos T Abel S ER-resident proteins PDR2 and LPR1 mediate thedevelopmental response of root meristems to phosphate availabilityProc Natl Acad Sci U S A 2009 10614174ndash14179

22 Aung K Lin SI Wu CC Huang YT Su CL Chiou TJ pho2 a phosphateoveraccumulator is caused by a nonsense mutation in a microRNA399target gene Plant Physiol 2006 1411000ndash1011

23 Duan K Yi K Dang L Huang H Wu W Wu P Characterization of asub-family of Arabidopsis genes with the SPX domain reveals their


diverse functions in plant tolerance to phosphorus starvation Plant J2008 54965ndash975

24 Cruz-Ramiacuterez A Oropeza-Aburto A Razo-Hernaacutendez F Ramiacuterez-Chaacutevez EHerrera-Estrella L Phospholipase DZ2 plays an important role in extra-plastidic galactolipid biosynthesis and phosphate recycling in Arabidopsisroots Proc Natl Acad Sci U S A 2006 1036765ndash6770

25 Misson J Raghothama KG Jain A Jouhet J Block MA Bligny R Ortet P Creff ASomerville S Rolland N Doumas P Nacry P Herrera-Estrella L Nussaume LThibaud MC A genome-wide transcriptional analysis using Arabidopsisthaliana Affymetrix gene chips determined plant responses to phosphatedeprivation Proc Natl Acad Sci U S A 2005 10211934ndash11939

26 Morcuende R Bari R Gibon Y Zheng W Pant BD Blaumlsing O Usadel BCzechowski T Udvardi MK Stitt M Scheible WR Genome-widereprogramming of metabolism and regulatory networks of Arabidopsisin response to phosphorus Plant Cell Environ 2007 3085ndash112

27 Calderoacuten-Vaacutezquez C Ibarra-Laclette E Caballero-Peacuterez J Herrera-Estrella LTranscript profiling of Zea mays roots reveals gene responses to phosphatedeficiency at the plant- and species-specific levels J Exp Bot 2008592479ndash2497

28 Nussaume L Kanno S Javot H Marin E Pochon N Ayadi A Nakanishi TMThibaud M-C Phosphate import in plants focus on the PHT1 transportersFront Plant Sci 2011 283

29 Chacoacuten-Loacutepez A Ibarra-Laclette E Saacutenchez-Calderoacuten L Gutieacuterrez-Alanis DHerrera-Estrella L Global expression pattern comparison between lowphosphorus insensitive 4 and WT Arabidopsis reveals an important roleof reactive oxygen species and jasmonic acid in the root tip responsePlant Signal Behav 2011 6(3)382ndash392

30 Ruepp A Zollner A Maier D Albermann K Hani J Mokrejs M Tetko IGuumlldener U Mannhaupt G Muumlnsterkoumltter M Mewes HW The FunCat afunctional annotation scheme for systematic classification of proteinsfrom whole genomes Nucleic Acids Res 2004 325539ndash5545

31 Nilsson L Muumlller R Nielsen TH Dissecting the plant transcriptome and theregulatory responses to phosphate deprivation Physiol Plant 2010139129ndash143

32 Cheng Y Zhou W El Sheery NI Peters C Li M Wang X Huang JCharacterization of the Arabidopsis glycerophosphodiesterphosphodiesterase (GDPD) family reveals a role of the plastid-localizedAtGDPD1 in maintaining cellular phosphate homeostasis under phosphatestarvation Plant J 2011 66781ndash795

33 Russo MA Quartacci MF Izzo R Belligno A Navari-Izzo F Long- and short-termphosphate deprivation in bean roots plasma membrane lipid alterations andtransient stimulation of phospholipases Phytochemistry 2007 681564ndash1571

34 Hammond JP Bennett MJ Bowen HC Broadley MR Eastwood DC May STRahn C Swarup R Woolaway KE White PJ Changes in gene expression inArabidopsis shoots during phosphate starvation and the potential fordeveloping smart plants Plant Physiol 2003 132578ndash596

35 Secco D Jabnoune M Walker H Shou H Wu P Poirier Y Whelana JSpatio-temporal transcript profiling of rice roots and shoots in responseto phosphate starvation and recovery Plant Cell 2013doihttpdxdoiorg101105tpc113117325

36 Bais HP Prithiviraj B Jha AK Ausubel FM Vivanco JM Mediation ofpathogen resistance by exudation of antimicrobials from roots Nature2005 434217ndash221

37 Millet YA Danna CH Clay NK Songnuan W Simon MD Werck-Reichhart DAusubel FM Innate immune responses activated in Arabidopsis roots bymicrobe-associated molecular patterns Plant Cell 2010 22973ndash990

38 Muchhal US Pardo JM Raghothama KG Phosphate transporters from thehigher plant Arabidopsis thaliana Proc Natl Acad Sci U S A 19969310519ndash10523

39 Brutnell TP Wang L Swartwood K Goldschmidt A Jackson D Zhu XGKellogg E Van Eck J Setaria viridis a model for C4 photosynthesisPlant Cell 2010 222537ndash2544

40 Malamy JE Benfey PN Organization and cell differentiation in lateralroots of Arabidopsis thaliana Development 1997 12433ndash44

doi1011861471-2229-14-69Cite this article as Alatorre-Cobos et al An improved low-cost hydro-ponic system for growing Arabidopsis and other plant species underaseptic conditions BMC Plant Biology 2014 1469

Submit your next manuscript to BioMed Centraland take full advantage of

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Abstract

Background

Results

Conclusions

Background

Results and discussion

Description of the hydroponic system

Protocol for setting up the hydroponic system

Applications of our hydroponic system 1) Quick transcriptional responses to Pi starvation

Arabidopsis growth and Pi-depletion responsive genes on Pi-starved hydroponic media

Exploring early genome-wide transcriptional responses to Pi depletion overview and functional classification of differentially expressed genes

Early transcriptional responses to low Pi availability involves cell wall modifications protein activity oxidation-reduction processes and hormones-mediated signaling that precede the reported Pi-signaling pathways

Applications of our hydroponic system 2) Pathological assays to evaluate systemic defense responses

Conclusions

Methods

Growth media

Plant material and growth conditions

Analysis of root architecture traits

GUS analyses

Microarray analysis

Transcript analysis

Pseudomonas syringae bioassays

Additional files

Abbreviations

Competing interests

Authorsrsquo contributions

Acknowledgements

Author details

References

Figure 1 Hydroponic system component dimensions andassembly A and B) Dimensions and assembly of seed-holder C)Assembled hydroponic system Containers with different volume forliquid media are shown The numbers at the bottomrsquos containerindicate the maximum volume and the number inside the containerthe volume of liquid media used in each case

Alatorre-Cobos et al BMC Plant Biology 2014 1469 of 13httpwwwbiomedcentralcom1471-22291469

but accessibility to the root system is then compro-mised Furthermore additional handling and thus unneces-sary plant stress during seedlings transfer to new growthmedia as well as during plant material collection shouldbe also considered when experiments on solid media aredesignedOne strategy for circumventing all problems described

above is the use of hydroponic systems for plant cul-ture Several hydroponic systems have been reported forArabidopsis [4-13] and some of them are now commer-cially available (Aeroponicsreg) [12] Most of these systemsare integrated by a plastic glass or polycarbonate containerwith a seed-holder constituted by rock wool a polyureth-ane (sponge) piece a steel or nylon mesh polyethylenegranulate or a polyvinyl chloride (PVC) piece Those areopen systems which allow axenic conditions or reducedalgal contamination into liquid growth media but sterilityis not possibleHere we describe step by step a protocol for setting up a

simple and low-cost hydroponic system that allows steril-ity conditions for growing Arabidopsis and other modelplants This new system is ideal for large-scale manipula-tion of seedlings and even for fully developed plants Oursystem is an improved version of Schlesier et al [8] inwhich the original glass jar and steel seed-holder are sub-stituted by a translucent polypropylene (PE) container anda piece of high-density polyethylene (HDPE) mesh Allcomponents are autoclavable reusable cheap and readilyavailable from local suppliers The new device designed asseed-holder avoids the use of low-melting agarose as sup-port for seeds allowing a quick and easy transfer to newmedia conditions andor harvest of plant material The ef-ficiency and functionality of our proposed system is dem-onstrated and exemplified in experiments that showedtypical early transcriptional changes under Pi starvationand pathogen infection

Results and discussionDescription of the hydroponic systemWe have improved a previously reported hydroponicsystem consisting of a glass jar and stainless piece inte-grated by a wire screen fixed between two flat rings andheld in place by three legs [8] by a simpler and cheapersystem assembled with a PE vessel and a seed-holder in-tegrated by a circle-shape HDPE mesh and two PE rings(Figure 1AB Table 1) Vessels and mesh used here arereadily available in local markets vessels are actuallyfood containers (Microgourmetreg Solo Cup USA wwwsolocupcom) available in food package stores while theHDPE mesh is a piece of anti-aphid mesh acquired inlocal stores providing greenhouse supplies (wwwtextilesagricolasmx) A small cotton plug-filled orifice in thecontainer lid allows gas exchange to the system (Figure 1C)This ventilation filter reduces but does not eliminate high

humidity in the medium container Such problem could besolved adding more ventilation filters or using other sealingmaterials as micropore 3 Mreg paper tape Aeration of the li-quid medium is not required for our hydroponic systemNo negative effects on plant growth have been observedwhen small tanks are used as medium containers (refer-ences in Table 1)The new seed-holder for positioning seeds on top of

the liquid medium consists of a mesh of HDPE monofil-aments held between two PE rings (ring A and B) withan area of 7854 cm2 (diameter =10 cm) which is able tohold 50 to 65 Arabidopsis seedlings for up to 10ndash15 daysafter germination (Figure 1AB Figure 2) (Table 1) Fullydeveloped Arabidopsis plants (2ndash3 plants per vessel) canalso be grown in this system if the container lid is replacedby another PE container (Additional file 1) Anti-aphidmesh with a 075 mm by 075 mm opening size (mesh usu-ally named 25 times 25) is adequate for keeping Arabidopsisseeds on top of the mesh (Figure 2AB) and allowing inde-pendent root system development (Figure 2CDE) Anti-aphid or anti-insect mesh with lower density can be usefulfor seeds larger than Arabidopsis seeds No legs for sup-porting the mesh-holder are needed in our hydroponicsystem The seed-holder is just placed into the container






photo slide mount

This system














References [3912] [41011] [6] [7] [5813]

Alatorre-C

oboset

alBMCPlant

Biology20141469

Page3of

13httpw

wwbiom


















4 Sterilization

























Time point sampled

10 min 30 min 2 h

Metabolism 967 159 214714 341241

Energy 322- 107- 243-


Transcription 129454 714214 121103

Protein fate 3221354 714714 121137








Cell fate -227 - -

































Additional files
































































Abstract

Background

Results

Conclusions

Background









Conclusions

Methods

Growth media



GUS analyses

Microarray analysis

Transcript analysis


Additional files

Abbreviations

Competing interests


Acknowledgements

Author details

References






photo slide mount

This system














References [3912] [41011] [6] [7] [5813]

Alatorre-C

oboset

alBMCPlant

Biology20141469

Page3of

13httpw

wwbiom


















4 Sterilization

























Time point sampled

10 min 30 min 2 h

Metabolism 967 159 214714 341241

Energy 322- 107- 243-


Transcription 129454 714214 121103

Protein fate 3221354 714714 121137








Cell fate -227 - -

































Additional files
































































Abstract

Background

Results

Conclusions

Background









Conclusions

Methods

Growth media



GUS analyses

Microarray analysis

Transcript analysis


Additional files

Abbreviations

Competing interests


Acknowledgements

Author details

References

















4 Sterilization

























Time point sampled

10 min 30 min 2 h

Metabolism 967 159 214714 341241

Energy 322- 107- 243-


Transcription 129454 714214 121103

Protein fate 3221354 714714 121137








Cell fate -227 - -

































Additional files
































































Abstract

Background

Results

Conclusions

Background









Conclusions

Methods

Growth media



GUS analyses

Microarray analysis

Transcript analysis


Additional files

Abbreviations

Competing interests


Acknowledgements

Author details

References



















Time point sampled

10 min 30 min 2 h

Metabolism 967 159 214714 341241

Energy 322- 107- 243-


Transcription 129454 714214 121103

Protein fate 3221354 714714 121137








Cell fate -227 - -

































Additional files
































































Abstract

Background

Results

Conclusions

Background









Conclusions

Methods

Growth media



GUS analyses

Microarray analysis

Transcript analysis


Additional files

Abbreviations

Competing interests


Acknowledgements

Author details

References






















Additional files
































































Abstract

Background

Results

Conclusions

Background









Conclusions

Methods

Growth media



GUS analyses

Microarray analysis

Transcript analysis


Additional files

Abbreviations

Competing interests


Acknowledgements

Author details

References





























Abstract

Background

Results

Conclusions

Background









Conclusions

Methods

Growth media



GUS analyses

Microarray analysis

Transcript analysis


Additional files

Abbreviations

Competing interests


Acknowledgements

Author details

References

methodology article open access an improved, low-cost

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