Il

MD

a

ARRAA

KAABCMS

1

sscbcp[o

dACsZ

T

0h

Plant Science 190 (2012) 40– 51

Contents lists available at SciVerse ScienceDirect

Plant Science

jou rn al hom epa ge: www.elsev ier .com/ locate /p lantsc i

n vitro shoot organogenesis and hormone response are affected by the alteredevels of Brassica napus meristem genes

ohamed Elhiti1, Claudio Stasolla ∗

epartment of Plant Science, University of Manitoba, Winnipeg, R3T 2N2 Manitoba, Canada

r t i c l e i n f o

rticle history:eceived 16 February 2012eceived in revised form 28 March 2012ccepted 1 April 2012vailable online 7 April 2012

eywords:uxinrabidopsisrassica napusytokinineristem

hoot organogenesis

a b s t r a c t

Arabidopsis shoot meristem activity is regulated by a molecular network involving the participationof several components, including SHOOTMERISTEMLESS (STM), CLAVATA1 (CLV1), and ZWILLE (ZLL). Inan effort to identify the role of these genes during in vitro shoot formation Brassica and Arabidopsisplants were transformed with the Brassica napus (Bn) STM, CLV1, ZLL1 and ZLL2 identified in previouswork [1]. In both systems shoot organogenesis was promoted by the over-expression of BnSTM, BnZLL1,and BnZLL2, and repressed by the over-expression of BnCLV1. This distinct regulation, analogous to thatoccurring during in vivo meristem formation where STM and ZLL encourage stem cell formation whileCLV1 accelerates transition to differentiation, suggests similar regulatory mechanisms governing shootformation in vivo and in vitro. While the BnZLL1 and BnZLL2 induction of shoot organogenesis corre-lated only to changes in auxin signaling, BnSTM and BnCLV1 evoked major transcriptional alterations incytokinin response. Besides increasing the transcript levels of two cytokinin receptors, ARABIDOPSIS HIS-TIDINE KINASE4 (AHK4) and CYTOKININ INDEPENDENT KINASE (CKI1), ectopic expression of BnSTM induced

Type-B ARABIDOPSIS RESPONSE REGULATORS (ARRs) and repressed Type-A ARRs. Opposite transcriptionalpatterns occurred in explants over-expressing BnCLV1, characterized by a decreased ability to produceshoots. The role played by Type-A and Type-B ARRs during shoot organogenesis was further examinedusing a genetic approach which revealed the requirement of ARR12 for the BnSTM positive regulationof shoot organogenesis. Collectively these results expand our knowledge on the function of meristemgenes, and provide new tools for enhancing in vitro propagation systems.

. Introduction

Shoot organogenesis refers to that process whereby de-novohoots are initiated from somatic cells of the explants. In culture,hoot formation can be executed directly, without an interveningallus phase, or indirectly, with a callus step. Examples of both haveeen documented [2]. Morphological, physiological, and geneticharacterizations of shoot organogenesis have identified distinct

hases: competence acquisition, canalization, and morphogenesis3]. In the first phase, cells within the explant acquire the abilityr competence to responds to inductive signals. These competent

Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; 2iP, 6-(�,�-imethylallylamino)-purine; AAR, ARABIDOPSIS RESPONSE REGULATOR; AHK,RABIDOPSIS HISTIDINE KINASE; CIM, callus induction medium; CLV1, CLAVATA1;KI1, CYTOKININ INDEPENDENT KINASE1; MDE, microspore-derived embryos; SIM,hoot induction medium; SAM, shoot apical meristem; STM, SHOOTMERISTEMLESS;LL, ZWILLE.∗ Corresponding author. Fax: +1 204 474 7528.

E-mail address: stasolla@ms.umanitoba.ca (C. Stasolla).1 Permanent address: Department of Botany, Faculty of Science, Tanta University,

anta 31527, Egypt.

168-9452/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.plantsci.2012.04.002

© 2012 Elsevier Ireland Ltd. All rights reserved.

cells are then “canalized” into the shoot developmental programwhich culminates in “morphogenesis”, that is the formation ofshoots. In Arabidopsis, viable shoots can be produced from severalexplants, including petals and sepal [4], hypocotyls [5] and roots[6]. In the latter case young roots are induced to form shoots bya procedure consisting of a preculture onto an auxin-containingmedium (callus induction medium, CIM), followed by a transferonto a cytokinin-containing medium (shoot induction medium,SIM) [6,7]. This efficient system has been very useful in examiningevents related to competence acquisition [7] and the requirementof specific factors needed for proper shoot formation [7–9].

Studies on primary hormone response during Arabidopsisorganogenesis revealed precise changes in the expression of genesinvolved in auxin and cytokinin signaling. Competence acquisitionrequires auxin which activates three distinct classes of gene fami-lies: Aux/IAA, GH3, and SAUR [10,11]. Specific Aux/IAA factors werein fact up-regulated during preincubation on the auxin-rich CIM[7]. Initiation of shoot formation on SIM is associated with profound

changes in cytokinin perception and signaling. Genetic dissection ofthe cytokinin pathway has identified several components, includ-ing sensor histidine kinases (AHKs), histidine phosphotransmitters(AHPs) and response regulators (ARRs). The Arabidopsis genome

ant Sc

corAceof[

igCeflofmtifSmf[

rddCtdCeacaabAgaZdhBWsaofi

2

2

(gsi(n1t

M. Elhiti, C. Stasolla / Pl

ontains ARRs which are grouped into two distinct groups basedn defined structural features. While the Type-A ARRs contain aeceiver domain and a very short C-terminal extension, Type-BRRs are characterized by a longer C-terminal extension whichontains regions acting as transcription regulators [12]. Geneticvidence indicates that Type-B ARRs are transcription activatorsf cytokinin-induced genes, including Type-A ARRs which act aseed-back repressors of the initial cytokinin transduction pathway13].

Cell pattern formation during shoot meristem developmentn vivo requires the dynamic expression of well characterizedenes. These include SHOOTMERISTEMLESS (STM), ZWILLE (ZLL),LAVATA1 (CLV1), and WUSCHEL (WUS) [14]. SHOOTMERISTEMLESSncodes a knotted-like homeobox protein present in vegetative andoral meristems and is required for the development of embry-nic meristems and their post-embryonic maintenance [15,16]. Theunction of STM is to suppress differentiation and maintain indeter-

inate cell fate within the SAM, as a mutation in this gene produceserminally differentiated cells in the apical pole [15]. Proper local-zation of STM is established by ZLL, a member of the ARGONAUTEamily, also required for the formation of the primary embryonicAM. However, unlike STM, ZLL is not needed for post-embryoniceristem function [17–19]. Adventitious meristems originating

rom fully differentiated SAMs were in fact observed in zll seedlings19].

An additional factor controlling the SAM is CLV1, a leucine-richepeat receptor kinase protein [20] responsible for promoting cellifferentiation of the meristematic cells by restricting the WUSomain through a complex feed-back mechanism involving otherLV members [21]. The balance between cell division and differen-iation is altered in clv1 plants which exhibit enlarged meristemsue to an atypical expansion of the WUS expression domain [22].LV1 acts antagonistically to STM, with the former limiting thexpansion of the undifferentiated stem cell population in the SAMnd promoting differentiation, and the latter maintaining stemells in an undifferentiated state [23]. This competitive functionlso operates during the specification of embryonic cells in vitro,s demonstrated by the differences in somatic embryos producedy Arabidopsis plants over-expressing the Brassica STM or CLV1 [1].s an extension of that work and to further characterize Brassicaenes involved in SAM formation, it is the objective of this study tonalyze the effects of altered expression of Brassica STM, CLV1 andLL during shoot organogenesis in Brassica and Arabidopsis, and toetermine whether the observed changes are due to alterations inormone response. The results indicate that ectopic expression ofnSTM and BnCLV1 have opposite effects on shoot organogenesis.hile BnSTM stimulates the process in both Brassica and Arabidop-

is, BnCLV1 has a repressive effect. This differential regulation isccompanied by precise transcriptional changes of key componentsf auxin and cytokinin signaling, including ARR12, which is identi-ed as a requirement for the BnSTM response.

. Materials and methods

.1. Plant material and transformation

Isolation of the Brassica napus (Bn) STM, CLV1, ZLL-1 and -2two ZLL clones were identified), ectopic expression of the fourenes in Arabidopsis, and transformation of B. napus with BnSTM inense and antisense orientation were conducted in previous stud-es [1]. Transformation of Brassica plants with the remaining genes

BnCLV1, BnZLL-1 and BnZLL2) was performed according to [24]. B.apus seeds were surface sterilized, and germinated for 5 days on/2 MS-B5 medium supplemented with 1% sucrose. Hypocotyl sec-ions (2–3 mm) were inoculated on co-cultivation medium (MS-B5

ience 190 (2012) 40– 51 41

medium supplemented with 3% sucrose and 2 mg/L BA) for 4 days,and then incubated with the Agrobacterium tumefaciens strainGV3101 harbouring the Gateway pK2GW7 (Invitrogen, USA, con-taining individual clones in sense orientation) or pK8WGIm24GW(containing individual clones for RNAi-mediated suppression). Fol-lowing a 4 day co-cultivation period, the explants were platedonto shoot induction medium (MS-B5 supplemented with 5 mg/LAgNO3, 2 mg/L BA, 3% sucrose, 20 mg/L timentin and 50 mg/Lkanamycin) for 8 weeks. The emerging shoots were first transferredonto elongation medium (MS-B5 supplemented with 3% sucrose,0.1 mg/L GA3, and 1 mg/L BA) for 4 weeks, and then placed on root-ing medium (MS-B5 supplemented with 1% sucrose, and 2 mg/LNAA) for 4 weeks. Rooted shoots were transferred onto soil togenerate fully mature plants (F0). Seeds from F0 plants were ger-minated on 1/2 MS basal medium with 1% sucrose and 50 mg/Lkanamicin and the resulting F1 generation was screened for thepresence of the transgene by RT-PCR using a forward 35S promoterprimer (5′-TGGACCCCCACCCACGAG-3′) and a reverse (for senseplants) gene specific primer (5′-AGACGATCCCCGGTCCGCCTTTG-3′ for BnCLV1, and 5′-CAGACTCTTTGTATAGTCTCACTA-3′ forBnZLL-1 and -2) or a forward (for RNAi plants) gene spe-cific primer (5′-TCTCCTTCAACGACCTCTCG-3′ for BnCLV1, and5′-GACGACGACGGCGGCAGCTCAGAGCC-3′ for BnZLL-1 and -2).Microspores harvested from the positive F1 plants were cultured toproduce haploid MDEs which were germinated on 1/2 MS mediumsupplemented with 1% sucrose and 70 mg/L kanamycin for 4 weeks.The haploid seedling were then treated with 0.2% colchicine for6 h in order to generate homozygous (double haploid) plants. Afterconfirming the presence of the transgene by RT-PCR, selected plantswere used as a source of seeds to generate the next (F2) generation.The expression level of the transgenes in these plants was measuredby quantitative (q) RT-PCR (primer sequences in SupplementalTable 1).

Seeds with T-DNA insertions within two Type-A ARRs (ARR4and 5) and three Type-B ARRs (ARR2, 10, and 12) were obtainedfrom the Arabidopsis Biological Research Center (Ohio State Uni-versity). The following seed stocks were utilized: arr4 (CS25266),arr5 (CS25267), arr2 (CS6947), arr10 (CS39989), and arr12 (CS6978).

2.2. Induction of shoot organogenesis

Shoot organogenesis from Arabidopsis root explants wasinduced exactly as described in Valvekens et al. [6]. Arabidop-sis seeds were sterilized for 30 s in 70% ethanol, 10 min in 0.67%NaOCl, rinsed three times in sterile water and plated on solidgermination medium (1/2 strength MS salts with 10 g/L sucroseand 6 g/L agar) for 1 week. Seedlings were transferred into liq-uid germination medium and incubated on a shaker at 100 rpmin the dark for another week. Roots were dissected from theseedlings and placed on a solid callus induction medium (CIM)containing MS salts and vitamins, 20 g/L glucose, 0.05 mg/L kinetin,and 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4D), and incubatedunder continuous light at 22 ◦C. After 4 days, the roots were trans-ferred on a solid shoot induction medium (SIM) containing MSsalts and vitamins, 20 g/L glucose, 0.15 mg/L IAA and 5 mg/L 6-(�,�-dimethylallylamino)-purine (2ip). After 14 days of incubationunder continuous light at 22 ◦C, shoots emerging from root tissuewere counted.

Shoot organogenesis in Brassica was initiated from hypocotylsections (2–3 mm) according to Bhalla and Singh [24]. After 8 weeksof incubation on SIM (MS-B5 medium supplemented with 3%

sucrose and 2 mg/L BA), the hypocotyl explants were transferredonto shoot elongation medium (MS-B5 supplemented with 3%sucrose, 0.1 mg/L GA3, and 1 mg/L BA) and the shoots were countedafter 3 weeks.

4 ant Sc

ba

2

KSs(eLTo(adoT

2

AttrdSw

2

tv

3

3i

aw[(2veoBBtd[cw

oteatB

2 M. Elhiti, C. Stasolla / Pl

Analyses of auxin and cytokinin requirements were performedy changing the levels of 2,4-D and 2iP in CIM and SIM, respectively,nd assessing the number of shoots formed.

.3. Gene expression studies

Total RNA was extracted using QIAGEN RNeasy® Plant Miniit (Cat. #74904) and used to generate cDNA with InvitrogenuperScript® II Reverse Transcriptase (Cat. #19064-002). Mea-urements of transcript levels were performed by quantitativeq)RT-PCR as described by Elhiti et al. [1]. The relative level of genexpression was analyzed with the 2−��CT method described byivak and Schmittgen [25] using actin (AY139999) as a reference.he selection of genes participating in hormone response was basedn the work of Che et al. [7] who identified 8 auxin responsive genesIAA1, 5, 8, 9, 11, 19, 20 and 29) which were up-regulated on CIM,nd several genes involved in cytokinin signaling which showedistinct expression profiles during the organogenic process. A listf primers utilized for the experiment is compiled in Supplementalable 1.

.4. RNA in situ hybridization studies

For RNA in situ hybridization studies the full length cDNA oftWUS was cloned into a pGEM Vector using pGEM-T Easy Vec-or System (Promega Cat. #A1360). The cDNA was amplified fromhe vector using T7 and SP6 primers and used for the prepa-ation of DIG-labeled sense and antisense riboprobes, exactly asescribed in the DIG Application Manual (Roche Applied Science).lide preparation, hybridization conditions, and color developmentere performed with the method outlined in Belmonte et al. [26].

.5. Statistical analysis

Unless specified, all experiments were performed using at leasthree biological replicates and the Tukey’s Post Hoc test for multipleariance [27] was used to compare differences among samples.

. Results

.1. Altered expression of Brassica genes affects shoot productionn culture

Expression levels of all the transgenes (BnSTM, BnCLV1, BnZLL1nd BnZLL2) in Arabidopsis plants, and of BnSTM in Brassica plantsere verified in previous studies (see Supplemental Figs. 1 and 2 in

1]). Down-regulation of BnCLV1 in Brassica via RNA-interferenceRNAi) resulted in the production of three lines: BnCLV1 (RNAi)-1,, and 3, which showed significant suppression of BnCLV1 in bothegetative and reproductive tissue (Supplemental Fig. 1). No regen-ration occurred from explants transformed with BnCLV1 in senserientation. Of the several plants ectopically expressing BnZLL1 andnZLL2, six were selected for this study: BnZLL1 (S-1, 2, and 3) andnZLL2 (S-1, 2, and 3). In all these lines the expression of the respec-ive transgene was induced in both leaves and flowers. The highegree of sequence similarity (>96%) between BnZLL1 and BnZLL21] allowed us to suppress both genes using a single BnZLL2 RNAionstruct. Three lines down-regulating both BnZLL1 and BnZLL2ere selected: BnZLL2 (RNAi)-1, 2, and 3 (Supplemental Fig. 1).

The altered expression of the Brassica genes affected the numberf shoots originating from Brassica explants (Fig. 1A). Shoot forma-ion from Brassica hypocotyl explants was stimulated by the ectopic

xpression of BnSTM (lines S15, 23, and 101), BnZLL1 (lines S1, 2,nd 3), and BnZLL2 (lines S1, 2, and 3) (Fig. 1A). A similar induc-ion was also observed in two lines with reduced expression ofnCLV1, BnCLV1-RNAi 1 and 3. A marginal decline in the number of

ience 190 (2012) 40– 51

shoots produced in culture occurred in two lines in which BnSTMwas suppressed via antisense transformation (BnSTM-A1 and 5)(Fig. 1A). To further asses if the effects of the four Brassica genes onthe organogenic process were retained in another species, BnSTM,BnCLV1, BnZLL1, and BnZLL2 were over-expressed in Arabidopsis. Asignificant increase in shoot formation occurred in Arabidopsis linesover-expressing BnSTM, especially in line 2, which showed a 7-foldincrease in shoot production relative to WT (Fig. 1B). Unlike WTtissue, where the organs emerged along the root surface as sepa-rate entities, clusters of shoots were often visible in explants withelevated BnSTM levels (Fig. 1C). A consistent, but less pronouncedincrease in shoot organogenesis was observed in lines ectopicallyexpressing BnZLL1 and BnZLL2, whereas a reduced ability to produceshoots occurred in lines with high levels of BnCLV1 (Fig. 1B).

Given the similar behavior exhibited by the transformed Brassicaand Arabidopsis explants in culture, we decided to further investi-gate the effects of BnSTM, BnZLL-1, BnZLL2 and BnCLV1 using theArabidopsis system which is more amenable to molecular studies.The following Arabidopsis lines were selected for the remainingexperiments: BnSTM-S2, BnZLL1-S2, BnZLL2-S4, and BnCLV1-S3.

3.2. Expression and localization of WUSCHEL in Arabidopsis linesover-expressing the Bn genes

The expression and localization patterns of WUSCHEL (WUS), areliable marker of shoot formation both in vivo [28] and in vitro[14], were analyzed in Arabidopsis during organogenesis. In theWT line WUS transcripts were induced on the cytokinin-rich shootinduction medium (SIM), and reached a maximum level at theend of the culture period (Fig. 2A). A similar profile, despite anearly induction on callus induction medium (CIM) and a higherexpression on SIM occurred in the explants of the BnSTM-S2 line.Over-expression of BnCLV1 decreased the abundance of WUS tran-scripts, which increased marginally in lines with elevated levels ofBnZLL1 and 2 upon transfer onto SIM (Fig. 2A). At day 4 on CIM,WUS was localized in distinct clusters of apical and a sub-apicalcells within the WT explants (Fig. 2B1). The localization of this genein explants ectopically expressing BnSTM was extended to a largerdomain (Fig. 2B2). This was in contrast to the BnCLV1-S3line, whichshowed a very restricted localization of WUS (Fig. 2B3). No differ-ences in WUS localization patterns were observed between the WTline and lines with increased levels of BnZLL1 or BnZLL2 (data notshown).

3.3. Auxin sensitivity and signaling during Arabidopsis shootorganogenesis

The requirement of the tissue for exogenously supplied auxin,which is needed for the acquisition of shoot competence [3], wasexamined by culturing the root explants on a CIM with differentlevels of 2,4-D. In WT tissue, shoot formation was halved when theconcentration of 2,4-D was reduced to 0.25 mg/L (Fig. 3). A moresevere reduction of this growth regulator significantly compro-mised shoot organogenesis as no shoots were observed at a 2,4-Dconcentration lower than 0.03 mg/L. An increased response to auxinwas observed in explants over-expressing BnSTM, which were stillable to produce 60% of shoots (compared to 20% of WT tissue) whenthe level of 2,4-D was lowered to 0.12 mg/L. In the BnSTM-S2 line,shoot production was still obtained at those auxin levels which pre-cluded morphogenesis in the WT line. A similar response to thatdescribed for the BnSTM over-expressing line was also observed

in the BnZLL1-S2 and BnZLL2-S4 lines. The introduction of BnCLV1decreased sensitivity to 2,4-D as no shoots were formed when theconcentration of this growth regulator was lower than 0.06 mg/L(Fig. 3).

M. Elhiti, C. Stasolla / Plant Science 190 (2012) 40– 51 43

Fig. 1. Effects of altered expression of Brassica napus (Bn) SHOOTMERISTEMLESS (STM), CLAVATA1 (CLV1), ZWILLE1 (ZLL1) and ZLL2 on the number of shoots produced inc nsgenl entagW shoo

aa8aoppialIoIel

ulture. (A) Number of shoots produced by hypocotyl explants of Brassica napus traines. Values + SE are means of three biological replicates and are expressed as perc

T (left panel) the over-expression of BnSTM (right panel) increased the clusters of

The expression levels of several Aux/IAA genes, involved in theuxin response during shoot formation in vitro [7] were measuredt different days in culture. As shown in Fig. 4, all genes (IAA1, 5,, 9, 11, 19, 20 and 29) were activated in WT tissue cultured on theuxin-rich CIM, and then repressed during the subsequent transfernto SIM. Among transformed lines several different expressionatterns were observed. While some genes (IAA 5 and 9) dis-layed similar expression profiles, others (IAA 8, 11, and 19) were

nduced on CIM especially in lines over-expressing BnSTM, BnZLL1,nd BnZLL2 (although the expression of IAA 19 in the BnZLL2-S4ine was not statistically different from WT). The expression ofAA 8, 11, and 19 was repressed in explants with elevated levels

f BnCLV1 (Fig. 4). Different expression profiles were observed forAA 20 which was induced only in the BnZLL1 and BnZLL2 over-xpressors, and IAA29 which was up-regulated in the BnSTM-S2ine (Fig. 4).

ic lines. (B) Number of shoots produced by root explants of Arabidopsis transgenice of WT. *Indicate values statistically different (p < 0.05) from WT. (C) Compared tots originating from Arabidopsis root explants.

3.4. Cytokinin sensitivity and signaling during Arabidopsis shootorganogenesis

The requirement for cytokinin during shoot organogenesis wasexamined by culturing the root explants on a SIM containing dif-ferent levels of the cytokinin 2iP. Shoot formation in WT tissuegradually declined with lower levels of 2ip (Fig. 5). A similarresponse was observed in lines over-expressing BnZLL1 and BnZLL2.The BnSTM-S2 line was still able to produce a substantial number ofshoots with low cytokinin levels (below 0.6 mg/L 2iP), whereas anopposite profile characterized the BnCLV1-S3 line where shoot for-mation was precluded at a 2iP concentration lower than 0.3 mg/L

(Fig. 5).

The expression profile of several genes previously described inrelation to shoot organogenesis [7] and encoding cytokinin recep-tors (AHK3, 4, and CKI1), Type-A (ARR 4, 5, 7, 15, and 16), and

44 M. Elhiti, C. Stasolla / Plant Science 190 (2012) 40– 51

Fig. 2. (A) Expression level of WUSCHEL (WUS) in transgenic Arabidopsis root explants over-expressing the Brassica napus (Bn) SHOOTMERISTEMLESS (STM), CLAVATA1 (CLV1),ZWILLE1 (ZLL1) and ZLL2. Measurements were conducted at specific days on callus induction medium (CIM) and shoot induction medium (SIM). Values + SE are means ofthree biological replicates and are expressed relative to the WT value at day 0 (set at 1). *Indicate values statistically different (p < 0.05) from WT at the same day in culture.S, sense. (B) Localization of WUS by RNA in situ hybridization at 4 days on CIM. Compared to WT (1), where the transcripts of WUS demarked separated clusters of cells(arrows), the expression of the gene in the BnSTM-S2 explants encompassed all the apicaexpressed WUS in the BnCLV1-S3 explants (3). No signal was detected in tissue hybridizedthree times with similar results.

0

20

40

60

80

100

120WT

BnSTM-S2

BnZLL1 -S2

BnZLL2 -S4

BnCLV1-S3

Percen

tage o

f sh

oots

0.5 0.25 0.12 0.06 0.03 0.01

2,4-D co ncentrat ion (mg/L )

Fig. 3. Effects of decreasing levels of the auxin 2,4-D on callus induction mediumon the number of shoots produced by transgenic Arabidopsis root explants over-expressing the Brassica napus (Bn) SHOOTMERISTEMLESS (STM), CLAVATA1 (CLV1),ZWILLE1 (ZLL1) and ZLL2. Shoots were counted after 18 days in culture (see Section 2).

l and sub-apical cells (arrow) (2). Only a few isolated small groups of cells (arrows) with sense probes (4). Scale bars = 30 �m. RNA in situ hybridization were repeated

Type-B response elements (ARR1, 2, 10, 12, and 13), was measuredin the transgenic lines. Of the three cytokinin receptors, AHK4 andCKI1 showed a pronounced activation at day 18 and 11 on SIM,respectively (Fig. 6). Compared to WT, both genes were highlyup-regulated in the line ectopically expressing BnSTM, with CKI1peaking at day 11 and AHK4 at day 18. Lower levels of AHK3, CKI1and AHK4 were generally detected in the BnCLV1-S3 line (Fig. 6).

Among type-A ARRs, ARR 4 and 16 showed small variations inexpression throughout the culture period (CIM + SIM) within eachtransformed line (Fig. 7). Higher levels of ARR 4 transcripts weremeasured in the BnCLV1-S3 line. The remaining ARRs (ARR5, 7, and15) were induced on SIM. Compared to WT, the ectopic expressionof BnSTM repressed (ARR5, 7, and 15), whereas an overall inductionof the three genes occurred in the BnCLV1-S3 line. No major differ-ences in the expression profiles of ARR5, 7, and 15 were observedbetween the WT line and lines with elevated levels of BnZLL1 andBnZLL2 (Fig. 7).

The expression levels of several Type-B ARRs were also measured

during shoot organogenesis. Within each line, the expression pro-file of ARR13 did not fluctuate during the culture period (Fig. 8). Anoverall induction of ARR1, 2, 10, and 12 occurred on SIM, especiallyin the line over-expressing BnSTM. The lowest transcript levels of

M. Elhiti, C. Stasolla / Plant Science 190 (2012) 40– 51 45

0

2

4

6

8

10

12

CIM SI M

Day 0 Day 4 Day 11 Day 18

IAA 1 WTBnSTM-S 2

BnCLV1-S 3

BnZLL1 -S2

BnZLL2 -S4

012345678

CIM SI M

Day 0 Day 4 Day 11 Day 18

IAA 8

0

2

4

6

8

10

12

CIM SI M

Day 0 Day 4 Day 11 Day 18

IAA 11

0

2

4

6

8

10

12

CIM SI M

Day 0 Day 4 Day 11 Day 18

IAA 20

Rela

tiv

e e

xp

ress

ion

012345678

CIM SI M

Day 0 Day 4 Day 11 Day 18

IAA 5

0

5

10

15

20

25

CIM SI M

Day 0 Day 4 Day 11 Day 18

IAA 9

0

5

10

15

20

25

CIM SI M

Day 0 Day 4 Day 11 Day 18

IAA 19

0

1

2

3

4

5

6

CIM SI M

Day 0 Day 4 Day 11 Day 18

IAA 29

*

*

*

*

*

**

* *

*

*

* *

* ** *

*

*

*

*

*

*

*

*

*

Fig. 4. Expression level of several auxin-responsive genes (IAA) in transgenic Arabidopsis root explants over-expressing the Brassica napus (Bn) SHOOTMERISTEMLESS (STM),C c daya at dad

Afia

3

iicT

LAVATA1 (CLV1), ZWILLE1 (ZLL1) and ZLL2. Measurements were conducted at specifire means of three biological replicates and are expressed relative to the WT valueay in culture. S, sense.

RR2, 10 and 12 were observed in the BnCLV1-S3 line. A simpli-ed diagram summarizing the temporal expression patterns of theuxin and cytokinin genes is shown in Fig. 9.

.5. Shoot organogenesis is affected by the expression of ARRs

Gene expression analysis revealed that while the BnSTM

mprovement of shoot organogenesis is accompanied by thenduction of Type-B ARRs, the inhibitory effect of BnCLV1 is asso-iated with the up-regulation of several Type-A ARRs (Fig. 9).o further examine the role played by the ARRs during the

s on callus induction medium (CIM) and shoot induction medium (SIM). Values + SEy 0 (set at 1). *Indicate values statistically different (p < 0.05) from WT at the same

organogenic process, we induced shoots from well characterizedarr mutant lines [29,30]. Shoot production was inhibited in T-DNAloss-of function Type-B arr2, 10, and 12 mutants, and induced inType-A arr4 and 5 explants (Fig. 10).

To further examine the interaction between BnSTM and ARR12(which is the most required ARR for shoot organogenesis, Fig. 10and is induced by BnSTM, Fig. 8) we introduced BnSTM into

the arr12 mutant background by crossing (Supplemental Fig. 2).The introduction of the BnSTM transgene was not able to res-cue the decrease in shoot formation caused by the mutation ofARR12 (Fig. 11).

46 M. Elhiti, C. Stasolla / Plant Sc

0

20

40

60

80

100

120

Percen

tage o

f sh

oots

5.0 2.5 1.2 0.6 0.3 0.15

2ip co ncentrat ion (mg/L )

WT

BnSTM-S2

BnZLL1 -S2

BnZLL2 -S4

BnCLV1-S3

Fig. 5. Effects of decreasing levels of the cytokinin 2ip on shoot induction mediumoeZ

4

aislBmoGt

Frda

n the number of shoots produced by transgenic Arabidopsis root explants over-xpressing the Brassica napus (Bn) SHOOTMERISTEMLESS (STM), CLAVATA1 (CLV1),WILLE1 (ZLL1) and ZLL2. Shoots were counted after 18 days in culture (see Section 2).

. Discussion

Organization and growth pattern of the SAM in vivo is medi-ted by the action of several components which through geneticnteractions regulate meristem homeostasis, i.e. the balance of divi-ion and differentiation of the meristematic cells [31]. Toward ourong term objective to identify molecular mechanisms controllingrassica meristem function during in vivo and in vitro develop-

ent, the present work documents the effects of altered expression

f BnSTM, BnCLV1, BnZLL1, and BnZLL2 on shoot organogenesis.enetic studies in Arabidopsis have explained the inner working of

he shoot meristem and the interaction between positive regulators

0

0.5

1

1.5

2

2.5

CIM SI M

Day 0 Day 4 Day 11 Day 18

AHK3

0

1

2

3

4

5

6

7

8

CIM

Day 0 Day 4 D

CKI1

Rel

ati

ve

exp

ress

ion

Rel

ati

ve

exp

ress

ion

**

* *

ig. 6. Expression level of three cytokinin receptors (ARABIDOPSIS HISTIDINE KINASE, AHoot explants over-expressing the Brassica napus (Bn) SHOOTMERISTEMLESS (STM), CLAVays on callus induction medium (CIM) and shoot induction medium (SIM). Values + SE at day 0 (set at 1). *Indicate values statistically different (p < 0.05) from WT at the same d

ience 190 (2012) 40– 51

required for the maintenance of stem cell indeterminacy (STM andZLL) and negative regulators accelerating transition toward differ-entiation (CLV1) [17–19,22,23]. Our data suggest that the functionof these genes might be retained during in vitro shoot formationwith BnSTM BnZLL1 and BnZLL2 encouraging shoot production andBnCLV1 repressing the organogenic process (Fig. 1). Furthermore,the similar responses obtained using two distinct culture systems(Brassica and Arabidopsis) and physiologically different explants(hypocotyls and roots) attest to the conserved nature of the molecu-lar mechanisms governing meristem formation. Of specific interestis not only the opposite response of explants transformed withBnSTM and BnCLV1, which is reminiscent of their competitive regu-lation in vivo [22,23], but also the mechanism through which thesegenes fulfill their function. The expression of WUS, which encodesa homeodomain-containing protein implicated in the formationof stem cells in vivo [23] and required for ectopic morphogene-sis in vitro [32], is antagonistically regulated by BnSTM and BnCLV1(Fig. 2). Accumulation and localization of WUS transcripts correlatewith the competence to produce shoots, possibly due to the abilityof this gene to enhance meristem fate acquisition and promote ded-ifferentiation [33]. A similar regulatory role of BnSTM and BnCLV1 onWUS expression was also observed during embryogenic cell forma-tion leading to the somatic embryogenesis [1]. These observationssuggest that the initial events characterizing shoot organogenesisand somatic embryogenesis share common molecular mechanismswhich regulate meristem formation in vivo.

Hormonal requirements during shoot regeneration in culturehave been investigated extensively, although the mechanismsthrough which hormones evoke morphogenic responses remain

unknown. In Arabidopsis the developmental events leading to theformation of shoots require two major events: acquisition of com-petence and shoot commitment [34]. Competence to responds toshoot formation signals is acquired on CIM and requires the auxin

0

1

2

3

4

5

6

7

CIM SI M

Day 0 Day 4 Day 11 Day 18

AHK4

SIM

ay 11 Day 18

*

*

*

WTBnSTM-S 2

BnCLV1-S3

BnZLL1 -S2

BnZLL2 -S4

Rel

ati

ve

exp

ress

ion

*

K3; AHK4; and CYTOKININ INDEPENDENT KINASE, CKI1) in transgenic ArabidopsisATA1 (CLV1), ZWILLE1 (ZLL1) and ZLL2. Measurements were conducted at specificre means of three biological replicates and are expressed relative to the WT value

ay in culture. S, sense.

M. Elhiti, C. Stasolla / Plant Science 190 (2012) 40– 51 47

0

1

2

3

4

5

CIM SI M

Day 0 Day 4 Day 11 Day 18

ARR4

Rel

ati

ve

exp

ress

ion

0

5

10

15

20

25

30

35

40

CIM SI M

Day 0 Day 4 Day 11 Day 18

ARR5

0

10

20

30

40

50

60

70

CIM SI M

Day 0 Day 4 Day 11 Day 18

ARR7

0

2

4

6

8

10

12

14

16

CIM SI M

Day 0 Day 4 Day 11 Day 18

ARR15

0

0.5

1

1.5

2

CIM SI M

Day 0 Day 4 Day 11 Day 18

ARR16

*

*

*

*

**

*

*

*

*

*

*

*

*

*

WTBnSTM-S 2

BnCLV1-S 3

BnZLL1 -S2

BnZLL2 -S4R

elati

ve

exp

ress

ion

Rel

ati

ve

exp

ress

ion

Rel

ati

ve

exp

ress

ion

Rel

ati

ve

exp

ress

ion

*

Fig. 7. Expression level of several Type-A ARABIDOPSIS RESPONSE REGULATORS (ARRs) in transgenic Arabidopsis root explants over-expressing the Brassica napus (Bn) SHOOT-MERISTEMLESS (STM), CLAVATA1 (CLV1), ZWILLE1 (ZLL1) and ZLL2. Measurements were conducted at specific days on callus induction medium (CIM) and shoot inductionm relatif

atovnws88maert(mawtrw

edium (SIM). Values + SE are means of three biological replicates and are expressedrom WT at the same day in culture. S, sense.

ctivation of several IAA genes [34,35]. The function of IAA pro-eins is to repress ARF function and modulate downstream eventsf auxin response [36]. Exposure to auxin and transcriptional acti-ation of IAAs is crucial for the acquisition of competence sinceo shoots are formed if explants are incubated directly on SIMithout a preculture period on CIM [7]. The increased expres-

ion level of many IAAs in explants over-expressing BnSTM (IAA1,, 11, 19, 29), and to a lesser extent BnZLL1 and BnZLL2 (IAA1,, 11, 20), suggests that the beneficial effects exercised by theseeristem-related genes on shoot organogenesis is mediated by

uxin signaling. This notion is supported by the repression of sev-ral IAAs in explants with elevated levels of BnCLV1 which are lessesponsive to shoot formation signals. The opposite expression pat-ern of IAAs between positive regulators of shoot organogenesisBnSTM, BnZLL1 and BnZLL2) and the negative regulator (BnCLV1)

ay be related to the different sensitivity to exogenously supplieduxin observed among transformed lines (Fig. 3). The mechanism

hereby auxin encourages the acquisition of competence in cul-

ure is poorly understood [37], although Su et al. [33] suggests aole for the PIN-induced auxin gradient in the activation of WUS,hich leads to the production of totipotent cells. The concomitant

ve to the WT value at day 0 (set at 1). *Indicate values statistically different (p < 0.05)

activation of IAAs and WUS in the BnSTM over-expressing line andtheir repression in the line with high BnCLV1 levels agree with thishypothesis.

Besides modulating signals related to competence acquisition,the altered expression of the Brassica meristem genes affectsthe cytokinin-driven shoot commitment step. Two of the threecytokinin receptors, AHK4 and CK1 are induced by BnSTM on SIM(Fig. 6). Described as the first cytokinin receptor, AHK4 is essen-tial for shoot regeneration in culture [38]. A similar requirement isalso fulfilled by CKI, which is sufficient to promote shoot develop-ment and cytokinin-responses in the absence of cytokinin [13,39].The BnSTM induction of both receptors might improve cytokininperception during the shoot commitment step, which explains theability of explants over-expressing BnSTM to produce shoots in alow cytokinin environment (Fig. 5). While the inductive role of STMon cytokinin biosynthesis through the up-regulation of ISOPEN-TENYL TRANSFERASE7 is well documented [40,41] its effects on

cytokinin receptors are new.

Downstream components of the cytokinin signaling includeARABIDOPSIS RESPONSE REGULATORS (ARRs) which are classifiedinto two separate functional categories: Type-A and Type-B. While

48 M. Elhiti, C. Stasolla / Plant Science 190 (2012) 40– 51

0

5

10

15

20

25

30

35

CIM SI M

Day 0 Day 4 Day 11 Day 18

ARR1

0

0.5

1

1.5

2

2.5

3

CIM SI M

Day 0 Day 4 Day 11 Day 18

ARR2

0

10

20

30

40

50

60

70

CIM SI M

Day 0 Day 4 Day 11 Day 18

ARR10

0

2

4

6

8

10

12

14

CIM SI M

Day 0 Day 4 Day 11 Day 18

ARR12

0

0.5

1

1.5

2

2.5

CIM SI M

Day 0 Day 4 Day 11 Day 18

ARR13

*

*

* *

*

*

*

* *

*

*

*

*

*

WTBnSTM-S 2

BnCLV1-S3

BnZLL1-S2

BnZLL2-S4R

elati

ve

exp

ress

ion

Rel

ati

ve

exp

ress

ion

Rel

ati

ve

exp

ress

ion

Rel

ati

ve

exp

ress

ion

Rel

ati

ve

exp

ress

ion

Fig. 8. Expression level of several Type-B ARABIDOPSIS RESPONSE REGULATORS (ARRs) in transgenic Arabidopsis root explants over-expressing the Brassica napus (Bn) SHOOT-MERISTEMLESS (STM), CLAVATA1 (CLV1), ZWILLE1 (ZLL1) and ZLL2. Measurements were conducted at specific days on callus induction medium (CIM) and shoot inductionm relatif

Tto7Stmsbt5wwwscp

edium (SIM). Values + SE are means of three biological replicates and are expressedrom WT at the same day in culture. S, sense.

ype-A ARRs act as feed-back repressors of the initial cytokininransduction pathway, Type-B ARRs are transcription activatorsf cytokinin-induced genes [13]. Several Type-A ARRs (ARR4, 5,, and 15) are repressed by BnSTM and induced by BnCLV1 onIM. Repression of these ARRs has been shown to be crucial forhe execution of morphogenic events. In arr4 and 5 Arabidopsis

utant plants, lateral root formation and elongation are moreensitive to cytokinin inhibition and these effects can be revertedy the introduction of the respective functional gene [42]. Specifico the present studies, explants with reduced levels of ARR4 or

were able to produce shoots in a low cytokinin environmenthich precluded shoot formation in WT tissue [42]. Similar effectsere also documented for the other two ARRs (ARR7 and 15)

hich are differentially regulated in our experiment. Inhibition of

hoot production and reduced sensitivity to exogenously appliedytokinin during root elongation were reported in Arabidopsislants over-expressing ARR15 [43]. In the same line, a repression of

ve to the WT value at day 0 (set at 1). *Indicate values statistically different (p < 0.05)

many cytokinin-activated components was caused by the ectopicexpression of ARR7 [9]. These observations strongly suggest thatthe opposite effect of BnSTM and BnCLV1 on shoot organogenesisis the result of the differential regulation of Type-A ARRs, which ispossibly mediated by the altered expression of WUS [44].

Among the Type-B ARRs, transcriptional activators of cytokininresponse, ARR1, 2, 10, and 12 were up-regulated by BnSTM anddown-regulated by BnCLV1 (Fig. 8). The requirement of these acti-vators during developmental processes triggered by cytokinin iswell established. The experimental induction of ARR2 was suffi-cient to promote shoot formation in the absence of cytokinin [12],while roots of arr12 plants were less sensitive to applications ofcytokinin during elongation assays [45]. The involvement of ARR12

during protoxylem differentiation [45] also suggests that expres-sion of this gene might be required during vascularization eventsoccurring during the formation of shoots. Taken together, theseresults show that acquisition of shoot commitment is facilitated

M. Elhiti, C. Stasolla / Plant Science 190 (2012) 40– 51 49

Fig. 9. Schematic diagram summarizing the expression profiles of auxin and cytokinin genes in transgenic Arabidopsis root explants cultured on callus induction medium( ) CytoF LL-1 as

baan

CIM) and shoot induction medium (SIM). (A) Auxin responsive genes (see Fig. 4). (Big. 7). Color in bars represents time and intensity of gene induction. Data for BnZimilar expression patterns.

y the repression of Type-A ARRs and induction of Type-B ARRs,nd that these transcriptional changes are modulated by BnSTMnd BnCLV1. Contrary to our expectations, BnZLL1 and BnZLL2 didot affect cytokinin signaling, suggesting that the improvement on

kinin receptor genes (see Fig. 5). (C) Type-A ARRs (see Fig. 6). (D) Type-B ARRs (seend -2 were summarized together as the over-expression of the two genes evoked

shoot organogenesis caused by their over-expression is merely dueto alterations of the auxin signaling.

The opposite role played by Type-A and Type-B ARRs on shootformation, was further demonstrated by analyzing the shoot

50 M. Elhiti, C. Stasolla / Plant Sc

0

50

100

150

200

250

300

WT 12 10 2 5 4

ARRs (Typ e-B) ARRs (Typ e-A )

Sh

oo

t p

rod

uct

ion

(% o

f W

T)

*

* *

*

*

Fig. 10. Shoot production in several ARABIDOPSIS RESPONSE REGULATORS (ARRs)kcd

pmAmr[rTtffiSc

raocsmmlp

Fkmv

nocked out mutant lines of Arabidopsis. Values + SE are means of three biologi-al replicates and are expressed as percentage of WT. *Indicate values statisticallyifferent (p < 0.05) from WT.

roduction capability of several T-DNA arr lines (Fig. 10). In agree-ent with the transcriptional studies, mutations of two Type-A

RRs analyzed (ARR4 and 5) increased shoot formation, whereasutations of Type-B ARRs had an opposite effect. Given the key

ole played by ARR12 in the cytokinin signaling pathways in roots45] (the explant used in our study), we examined if this gene wasequired for the BnSTM improvement of the embryogenic process.he inability of BnSTM to rescue the poor organogenic output ofhe arr12 line (Fig. 11) is indicative of the requirement of ARR12or the BnSTM-shoot production. To our knowledge this is therst report documenting a possible genetic interaction betweenTM and ARR12, which might contribute to the STM regulation ofytokinin activity [40].

In conclusion, this work provides compelling evidence for theole played by four meristem Brassica genes, BnSTM, BnCLV1, BnZLL1nd BnZLL2, in the control of shoot organogenesis in vitro. Thepposite effect of BnSTM and BnCLV1 on shoot production, remines-ent of their antagonistic roles during the in vivo regulation of thehoot apical meristem, is a clear indication that similar molecularechanisms govern in vivo and in vitro shoot formation. Further-

ore, the different behavior in culture observed in the transformed

ines is mediated by the differential regulation of componentsarticipating in auxin and cytokinin response. While BnZLL1 and

0

20

40

60

80

100

120

Sh

oo

t p

rod

uct

ion

(% o

f W

T)

WT arr12 BnS TM / arr12

*

*

ig. 11. Shoot production in the Arabidopsis arr12 knock-out line and in an arr12nock-out line in which BnSTM was over-expressed (BnSTM/arr12). Values + SE areeans of three biological replicates and are expressed as percentage of WT. *Indicate

alues statistically different (p < 0.05) from WT.

[

[

[

[

[

[

[

[

[

[

ience 190 (2012) 40– 51

BnZLL2 appear to influence only the auxin-induced competencestep on CIM, BnSTM and BnCLV1 also affect the shoot commitmentresponse on SIM by regulating genes of cytokinin perception andsignaling. Through an overall repression of Type-A ARRs and theinduction of Type-B ARRs, BnSTM encourages shoot initiation. Inthis regulatory network ARR12 is required in the BnSTM response.Besides expanding knowledge on the function of meristem genes,this study provides new tools for enhancing in vitro propagationsystems.

Acknowledgment

This work was supported by a NSERC Discovery Grant to CS.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.plantsci.2012.04.002.

References

[1] M. Elhiti, M. Tahir, R.H. Gulden, K. Khamiss, C. Stasolla, Modulation of embryo-forming capacity in culture through the expression of Brassica genes involvedin the regulation of the shoot apical meristem, Journal of Experimental Botany61 (2010) 4069–4085.

[2] G. Hicks, Shoot induction and organogenesis in vitro: a developmental perspec-tive, In Vitro Cellular & Developmental Biology 30 (1994) 10–15.

[3] M. Sugiyama, Genetic analysis of plant morphogenesis in vitro, InternationalReview of Cytology 196 (2000) 67–84.

[4] S. Tanaka, Formation of callus and redifferentiation of shoots from Arabidopsisthaliana petal and sepal explants, Natural Science and Applied Science 49 (2001)277–283.

[5] J. Campos-Cuevas, R. Pelagio-Flores, J. Gonzalez-Raya, A. Mendez-Bravo, R.Ortiz-Castro, J. Lopez-Bucio, Tissue culture of Arabidopsis thaliana explantsreveal a stimulatory effect of alkamides on adventitious root formation andnitric oxide accumulation, Plant Science 174 (2008) 165–173.

[6] D. Valvekens, M. Montagu, M. Lijsebettebs, Agrobacterium tumefaciens medi-ated transformation of Arabidopsis thaliana root explants by using kanamycinselection, Proceedings of the National Academy of Sciences of the United Statesof America 85 (1988) 5536–5540.

[7] P. Che, D. Gingerich, S. Lall, S. Howell, Global and hormone-induced geneexpression changes during shoot development in Arabidopsis, Plant Cell 14(2002) 2271–2279.

[8] S. Ozawa, I. Yasutani, H. Fukuda, A. Komamine, M. Sugiyama, Organogenicresponses in tissue culture of srd mutants of Arabidopsis thaliana, Development125 (1998) 135–142.

[9] D. Lee, S. Kim, Y.M. Ha, J. Kim, Phosphorylation of Arabidopsis response reg-ulator 7 (ARR7) at the putative phospho-accepting site is required for ARR7to act as a negative regulator of cytokinin signalling, Planta 227 (2007)577–583.

10] S. Abel, A. Theologis, Early genes and auxin action, Plant Physiology 111 (1996)9–17.

11] T. Berleth, N.T. Krogan, E. Scarpella, Auxin signals: turning genes on and turningcells around, Current Opinion in Plant Biology 7 (2004) 553–563.

12] H. Sakai, T. Aoyama, A. Oka, Arabidopsis ARR1 and ARR2 response regulatorsoperate as transcriptional activators, Plant Journal 24 (2000) 703–711.

13] I. Hwang, J. Sheen, Two-component circuitry in Arabidopsis cytokinin signaltransduction, Nature 413 (2001) 383–389.

14] S.P. Gordon, M.G. Heisler, G. Reddy, C. Ohno, P. Das, E.M. Meyerowitz, Pat-tern formation during de novo assembly of the Arabidopsis shoot meristem,Development 134 (2007) 3539–3548.

15] M.K. Barton, R.S. Poethig, Formation of the shoot apical meristem in Arabidopsisthaliana: an analysis of development in the wild type and in the shootmeristem-less mutant, Development 119 (1993) 823–831.

16] K. Endrizzi, B. Moussian, A. Haecker, J.Z. Levin, T. Laux, The SHOOTMERISTEMLESSgene is required for maintenance of undifferentiated cells in Arabidopsis shootand floral meristems and acts at a different regulatory level than the meristemgenes WUSCHEL and ZWILLE, Plant Journal 10 (1996) 967–979.

17] J.R. McConnell, M.K. Barton, Effect of mutations in the PINHEAD gene of Ara-bidopsis on the formation of the shoot apical meristem, Developmental Genetics16 (1995) 358–366.

18] B. Moussian, H. Schoof, A. Haecker, G. Jürgens, T. Laux, Role of ZWILLE gene inthe regulation of central shoot meristem cell fate during Arabidopsis embryo-

genesis, EMBO Journal 17 (1998) 1799–1809.

19] K. Lynn, A. Fernandez, M. Aida, J. Sedbrook, M. Tasaka, P. Masson, M.K. Bar-ton, The PINHEAD/ZWILLE gene acts pleiotropically in Arabidopsis developmentand has overlapping functions with the ARGONAUTE1 gene, Development 126(1999) 469–481.

ant Sc

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

M. Elhiti, C. Stasolla / Pl

20] S.E. Clark, R.W. Williams, E.M. Meyerowitz, The CLAVATA 1 gene encodes a puta-tive receptor kinase that controls shoot and floral meristem size in Arabidopsis,Cell 89 (1997) 575–585.

21] S. Dodsworth, A diverse and intricate signalling network regulates stem cellfate in the shoot apical meristem, Developmental Biology 336 (2009) 1–9.

22] H. Schoof, M. Lenhard, A. Haecker, K.F. Meyer, G. Jürgens, T. Laux, The stem cellpopulation of Arabidopsis shoot meristems is maintained by a regulatory loopbetween CLAVATA and WUSCHEL genes, Cell 100 (2000) 635–644.

23] S.E. Clark, S.E. Jacobsen, J.Z. Levin, E.M. Meyerowitz, The CLAVATA and SHOOT-MERISTEMLESS loci competitively regulate meristem activity in Arabidopsis,Development 122 (1996) 1567–1575.

24] P.L. Bhalla, M.B. Singh, Agrobacterium-mediated transformation of Brassicanapus and Brassica oleracea, Nature Protocols 3 (2008) 181–189.

25] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2-Delta Delta C(T), Methods 25 (2001) 402–408.

26] M. Belmonte, A.J. Ambrose, A.R.S. Ross, S.R. Abrams, C. Stasolla, Improveddevelopment of microspore derived embryo cultures of Brassica napus cvTopaz following changes in glutathione metabolism, Physiologia Plantarum127 (2006) 690–700.

27] J.H. Zar, Biostatistical Analysis, 4th ed., Prentice-Hall, Englewood Cliff, 1999,208–228.

28] K.F. Mayer, H. Schoof, A. Haecker, M. Lenhard, G. Jürgens, T. Laux, Role ofWUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem, Cell95 (1998) 805–815.

29] J.P.C. To, G. Haberer, F.J. Ferreira, J. Deruère, M.G. Mason, G.E. Schaller, J.M.Alonso, J.R. Ecker, J.J. Kieber, Type-A, ARRs are partially redundant negativeregulators of cytokinin signaling in Arabidopsis, Plant Cell 16 (2004) 658–671.

30] M.G. Mason, D.E. Mathews, D.A. Argyros, B.B. Maxwell, J.J. Kieber, J.M. Alonso,J.R.S. Ecker, G.E. Shaller, Multiple type-B response regulators mediate cytokininsignal transduction in Arabidopsis, Plant Cell 17 (2005) 3007–3018.

31] M.K. Barton, Twenty years on: the inner working of the shoot apical meristem,a developmental dynamo, Developmental Biology 341 (2010) 95–113.

32] J.R. Zuo, Q.W. Niu, G. Frugis, N.H. Chua, The WUSCHEL gene promotes vegetative-to-embryonic transition in Arabidopsis, Plant Journal 30 (2002) 349–359.

33] Y.H. Su, X.Y. Zhao, Y.B. Liu, C.L. Zhang, S. O’Neill, Z.S. Zhang, Auxin-inducedWUS expression is essential for embryonic stem cell renewal during somaticembryogenesis in Arabidopsis, Plant Journal 59 (2009) 448–460.

[

ience 190 (2012) 40– 51 51

34] A.J. Cary, P. Che, S.H. Howell, Development events and shoot meristem geneexpression patterns during shoot development in Arabidopsis thaliana, PlantJournal 32 (2002) 867–876.

35] E. Liscum, J.W. Reed, Genetics of Aux/IAA and ARF action in plant growth anddevelopment, Plant Molecular Biology 49 (2002) 387–400.

36] T. Ulmasov, J. Murfett, G. Hagen, T.J. Guilfoyle, Aux/IAA proteins repress expres-sion of reporter genes containing natural and highly active synthetic auxinresponse elements, Plant Cell 9 (1997) 1963–1971.

37] V. Raghavan, Role of 2,4-dichlorophenoxyacetic acid (2,4-D) in somaticembryogenesis on cultured zygotic embryos of Arabidopsis: cell expansion, cellcycling, and morphogenesis during continuous exposure of embryos to 2,4-D,American Journal of Botany 91 (2004) 1743–1756.

38] T. Inoue, M. Higuchi, Y. Hashimoto, M. Seki, M. Kobayashi, T. Kato, S. Tabata,K. Shinozaki, T. Kakimoto, Identification of CRE1 as a cytokinin receptor fromArabidopsis, Nature 409 (2001) 1060–1063.

39] T. Kakimoto, CKI1, a histidine kinase homolog implicated in cytokinin signaltransduction, Science 274 (1996) 982–985.

40] S. Jasinski, P. Piazza, J. Craft, A. Hayo, L. Woolley, L. Rien, A. Phillips, P. Hed-den, M. Tsiantis, KNOX action in Arabidopsis is mediated by the coordinatedregulation of cytokinin and gibberellin activities, Current Biology 15 (2005)1560–1565.

41] O. Yanai, E. Shani, K. Dolezal, P. Tarkowski, R. Sablowski, G. Sanderberg, A.Samach, N. Ori, Arabidopsis KNOXI proteins activate cytokinin biosynthesis,Current Biology 15 (2005) 1566–1571.

42] J.P.C. To, J.J. Kieber, Cytokinin signalling: two components and more, Trends inPlant Science 13 (2008) 8591–8594.

43] T. Kiba, H. Yamada, S. Sato, T. Kato, S. Tabata, T. Yamashino, T. Mizuno, TheType-A response regulator ARR15 acts as a negative regulator in the cytokinin-mediated signal transduction in Arabidopsis thaliana, Plant and Cell Physiology44 (2003) 868–874.

44] A. Leibfried, J.P.C. To, W. Busch, S. Stehling, A. Kehle, M. Demar, J.J. Kieber,J.U. Lohmann, WUSCHEL controls meristem function by direct regulation of

cytokinin-inducible response regulators, Nature 438 (2005) 1172–1175.

45] A. Yokoyama, T. Yamashino, Y.-I. Amano, Y. Tajima, A. Imamura, H. Sakakibara,T. Mizuno, Type-B ARR transcription factors,ARR10 and ARR12 are implicatedin cytokinin-mediated regulation of protoxylem differentiation in roots of Ara-bidopsis thaliana, Plant and Cell Physiology 48 (2007) 84–96.