A comparative analysis of the Arabidopsis mutant amp1-1 and a novel weak amp1 allele reveals new functions of the AMP1 protein

A comparative analysis of the Arabidopsis mutant amp1-1 and a novel weak amp1 allele reveals new functions of the AMP1 protein
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  Planta (2007) 225:831–842 DOI 10.1007/s00425-006-0395-9  1 3 ORIGINAL ARTICLE A comparative analysis of the  Arabidopsis  mutant amp1-1  and a novel weak amp1  allele reveals new functions of the AMP1 protein Nelson J. M. Saibo · Wim H. Vriezen · Liesbeth De Grauwe · Abdelkrim Azmi · Els Prinsen · Dominique Van Der Straeten Received: 22 April 2006 / Accepted: 22 August 2006   / Published online: 28 September 2006 ©  Springer-Verlag 2006 Abstract Ethylene and gibberellins have a synergisticstimulatory e V  ect on hypocotyl elongation of light-grown  Arabidopsis thaliana  (L.) Heynh. seedlings. Ascreen for mutants with decreased response to thesehormones led to the isolation of a novel allele ( amp1-7  )of the  ALTERED MERISTEM PROGRAM   (  AMP  ) 1 locus. The amp1-7   allele contains a missense mutationcausing a phenotype, which is weaker than that of the amp1-1  mutant that carries a nonsense mutation. Themutant phenotype prompted the hypothesis thatAMP1 is involved in ethylene and GA signallingpathways or in a parallel pathway-controlling cell andhypocotyl elongation and cellular organization.  Amp1 mutants contain higher zeatin concentrations causingenlargement of the apical meristem, which was con- W rmed by cytokinin application to wild type seedlings.Light grown amp1  seedlings have shorter hypocotylsthan wild type; however, application of cytokininspromotes hypocotyl elongation of both Col-0 and amp1 . We suggest that in amp1  mutants either zeatinoverproduction or its action is strictly localized. Keywords Cytokinins · Ethylene · Gibberellins · Hypocotyl elongation · Shoot apical meristem · Zeatin Abbreviations ACC1-Aminocyclopropane-1-carboxylic acidAMPAltered meristem programCKCytokininsGAGibberellinsGA 3 Gibberellic acidLNMLow-nutrient mediumSAMShoot apical meristem Introduction Plant hormones regulate growth and developmentthroughout the plant life cycle. The e V  ect of phytohor-mones on growth is known to be tissue dependent as wellas regulated by environmental conditions (Davies 1995).Hypocotyl growth of  Arabidopsis thaliana  has beenextensively used as a model to study hormonal e V  ects onelongation and to investigate hormonal interactions. Inlight grown seedlings, gibberellins (GAs) enhance hypo-cotyl elongation through regulation of cell elongation(Cowling and Harberd 1999). In contrast, GAs do not Nelson J. M. Saibo and Wim H. Vriezen contributed equally to this work.N. J. M. Saibo · W. H. Vriezen · L. De Grauwe · D. Van Der Straeten ( & )Unit Plant Hormone Signaling and Bio-imaging, Department of Molecular Genetics, Ghent University, K.L. Ledeganckstraat 35, 9000 Gent, Belgiume-mail: dominique.vanderstraeten@ugent.beA. Azmi · E. PrinsenLaboratory of Plant Biochemistry and Physiology, Department of Biology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium Present Address: N. J. M. SaiboPlant Genetic Engineering Laboratory, Instituto de Tecnologia Química e Biológica, Av. da República—Quinta do Marquês, 2784-505 Oeiras, Portugal Present Address: W. H. VriezenDepartment of Plant Cell Biology, Radboud University Nijmegen, Toernooiveld 1, 6525 Nijmegen, The Netherlands  832Planta (2007) 225:831–842  1 3 show any e V  ect on the elongation of etiolated hypocotyls,suggesting that in these conditions GA levels saturate theGA response (Cowling and Harberd 1999). Ethylene caninhibit (Abeles etal. 1992) or stimulate (Smalle etal.1997) hypocotyl elongation depending on whether seed-lings are grown under dark or light conditions, respec-tively. We have reported that ethylene and GAs actsynergistically in promoting hypocotyl elongation of seedlings that are grown on low-nutrient medium (LNM)in the light (Saibo etal. 2003). These W ndings indicatethat ethylene and gibberellin response pathways mayinteract to modulate hypocotyl elongation.Understanding of the e V  ect of cytokinins (CK) inhypocotyls of light grown seedlings is rather limited.Benzyladenine (a synthetic cytokinin) does not a V  ecthypocotyl elongation of light grown  Arabidopsis  seed-lings (Su and Howell 1995), unless ethylene action isblocked (Smets etal. 2005). In dark-grown  A. thaliana seedlings CK inhibit hypocotyl elongation, an e V  ectthat is largely mediated by ethylene (Cary etal. 1995;Su and Howell 1995; Golan etal. 1996). The CK are known to stimulate ethylene production (Mattoo andWhite 1991) by activating ACC synthase, enzymes thatcontrol ethylene production under normal conditions(Vogel etal. 1998; Chae etal. 2003). In contrast, dark- grown seedlings treated with high levels of zeatin showa de-etiolation response: short hypocotyl, open cotyle-dons and no apical hook. The latter feature is exagger-ated by ethylene treatment (ChinAtkins etal. 1996).Cytokinins also play a role in the development of theshoot apical meristem (SAM) (Howell etal. 2003). Plantsover-producing CK show increased expression levels of  KNOTTED  ( KN1 ) homeobox ( KNOX  ) genes (Ruppetal. 1999). Conversely, overexpression of KNOX   genesleads to higher CK levels (Hewelt etal. 2000; Frugis etal.2001; Yanai etal. 2005). It has been shown that CK medi- ate KNOX action (Jasinski etal. 2005; Yanai etal. 2005). Some of the KN1  genes are expressed exclusively in theSAM and are involved in its development and mainte-nance (Jackson etal. 1994; Kerstetter etal. 1994; Kerstet- ter and Hake 1997). An other homeodomain gene WUSCHEL  ( WUS ) is also important for meristem regu-lation (Leyser 2003). WUS is a positive regulator of stemcells that directly represses the transcription of severaltwo-component ARABIDOPSIS RESPONSE REGU-LATOR (ARR) genes which act in the negative feed-back loop of cytokinin signalling (Kiba etal. 2003; Toetal. 2004). ARR genes might negatively in X uence meri-stem size and their repression by WUS might be neces-sary for proper meristem function (Leibfried etal. 2005).In addition to CK, GAs may also play a role in SAMdevelopment. Altered GA levels were observed inplants overexpressing KNOX   genes (Kusaba etal.1998) and it was demonstrated that the KNOX proteinNTH15 represses a GA biosynthesis gene (Sakamotoetal. 2001). An antagonistic e V  ect between ethyleneand the KNOX   gene KNAT2  was described, indicatingthat ethylene is also involved in SAM development(Hamant etal. 2002). It was proposed that ethyleneand CK act antagonistically in the meristem viaKNAT2 to regulate meristem activity.The amp1-1  ( altered meristem program ) mutant of   Arabidopsis  was reported to produce higher levels of CK as compared to wild type (Chaudhury etal. 1993;ChinAtkins etal. 1996). The amp1-1  mutant is charac-terized by an increased rate of leaf initiation, polyco-tyly, reduced elongation of the hypocotyl, de-etiolationin dark-grown plants, increased branching, an enlargedapical shoot meristem, semi-sterility and early X ower-ing (Chaudhury etal. 1993; Mordhorst etal. 1998). The  AMP1  gene encodes a putative glutamate carboxypep-tidase, possibly involved in peptide signalling (Helli-well etal. 2001). In addition to amp1-1 , amp1-2  and amp1-3 , other alleles of amp1  have been independentlyisolated and called  primordial timing  (  pt  ; Mordhorstetal. 1998), constitutively photomorphogenic  ( cop2 ;Hou etal. 1993) and hauptling  ( hpt  ; Mayer etal. 1991).In this study, we have isolated a new amp1  allelethat was called amp1-7  . This mutant was isolated basedon a screen designed to isolate mutants with adecreased response of the hypocotyl to 1-aminocyclo-propane-1-carboxylic acid (ACC)+Gibberellic acid(GA 3 ), a treatment that strongly induces hypocotylelongation in the light. Material and methods Plant material and growth conditions  Arabidopsis thaliana  (L.) Heynh. (ecotype Columbia)seeds were purchased from Lehle Seeds (Round Rock,TX, USA). The  Arabidopsis  mutants hls1  and amp1-1 ,both in Columbia background were obtained from the  Arabidopsis  Biological Resource Centre at Ohio StateUniversity. The GA 3 , ACC and zeatin were purchasedfrom Sigma-Aldrich (St. Louis, MO, USA).  Arabidop- sis  seeds were surface sterilized by incubation for15min in 5% (v/v) sodium hypochlorite plus 0.1%(v/v) Tween 20 and subsequently washed with sterile,distilled water. Sterile seeds were sown on LNM(Smalle etal. 1997) supplemented with hormones(ACC, GA 3  and zeatin) and allowed to imbibe at 4°Cfor 2days before incubation in a growth chamber at22°C and 60% relative humidity under white X uores-cent light [photosynthetic photon X ux density (PPFD):  Planta (2007) 225:831–842833  1 3 75   molm ¡ 2 s ¡ 1 ] and long-day conditions (16h light/8h dark). When  Arabidopsis  was grown for more than9days or for cytokinin quanti W cation (7-day-old seed-lings), seeds were sown on half-strength Murashige andSkoog (MS/2; Sigma) supplemented with 1% sucrose.Hypocotyls from 9-day-old seedlings were measured aspreviously described (Saibo etal. 2003).Isolation of mutantsFor mutant screening, M2 seeds of  A. thaliana  ecotypeColumbia mutagenised with ethyl methane sulfonate(EMS) were purchased from Lehle Seeds (RoundRock, TX, USA). From a population of 5,000 9-day-oldseedlings grown on LNM supplemented with 50   MACC+10   M GA 3 , 32 mutant candidates were iso-lated, based on their short hypocotyl, comparable withthat of non-treated seedlings. To reduce the number of false positives, seeds that had not germinated on daythree after imbibition were discarded. The F1 of those32 candidates was reselected on LNM+ACC+GA 3 and only 2 [  ACC + GA  3  insensitive 5  ( agi5 ) and agi 16 ]proved to be truly positives.Segregation analysisBoth mutants, agi5  and agi16 , were backcrossed withCol-0. Given that both carry recessive mutations, theirhomozygous mutants were selected directly from theF2. Homozygous agi5  were selected based on theirshort hypocotyl when grown on ACC+GA 3 , whereas,homozygous agi16  were selected on their hookless phe-notype. To test possible allelism, agi16  was crossedwith both amp1-1  and hls1 . In addition, agi16  wascrossed with Landsberg erecta  to generate a mappingpopulation. From the cross between agi16  and Lands-berg, 83 of 379 F2 seedlings were scored as homozy-gous for agi16 . DNA was isolated from a single leaf of these 83 individuals using a single step protocol(Thomson and Henry 1995). Mapping of the agi16 locus was performed using simple sequence lengthpolymorphism (SSLP) markers (Bell and Ecker 1994).Microscopy analysisFor confocal microscopy analysis, 8-day-old light-grown seedlings were W xed for 2h, with gentle agita-tion, in a solution prepared as follows: W ve volumes(vol) propionic acid +10 vol 37% formaldehyde +70 vol100% ethanol, and subsequently kept in 70% ethanolat 4°C (seedlings can be kept at this stage for severalweeks). After rehydration in a graded ethanol seriesfor 30min at each step, seedlings were treated with20   gml ¡ 1  RNase in 500mM NaCl, 10mM Tris–HCl,1mM EDTA; pH 7.4 for 30min at 37°C. After rinsingwith incubation bu V  er, seedlings were incubated for2days at 4°C in 0.1mM arginine pH 12.4 containing5   gml ¡ 1  propidium iodide. Subsequently, seedlingswere washed twice for 30min in 0.1M arginine pH 8and mounted in the same bu V  er. The analysis of theSAM after propidium iodide stain was done with aninverted confocal laser-scanning microscope LSM510(Zeiss) W tted with a X20 plan-apochromat objective.The number of cells in the L1 layer of the SAM wasdetermined by counting the number of nuclei in theoptical section that crosses the centre of the SAM (sec-tion that shows the highest number of L1 cells).Di V  erential contrast (DIC) microscopy was per-formed with 4-day-old dark-grown seedlings treated aspreviously described (Saibo etal. 2003).Extraction and puri W cation of cytokinins  Arabidopsis  seedlings were ground in liquid nitrogen,transferred into Bieleski solution (Bieleski 1964), andextracted overnight at ¡ 20°C. Before centrifugation(20,000  g , for 15min at 4°C), 5pmol of deuterated CKwere added to the samples as internal standards ( 2 H 3 -DHZ, 2 H 3 -DHZR, 2 H 3 -DHZR-P, 2 H 5 -Z-N 9 -G, 2 H 6 -IP, 2 H 6 -IPA, 2 H 6 -IP-N 9 -G and 2 H 6 -IPA-P; OlchemIm,Czech Republic). The supernatant was puri W ed W rst ona combination of DEAE-Sephadex and RP-C18 car-tridges and then secondly into immunoa Y nity columnscontaining immobilized antibodies against CK (OlChe-mIm, Czech Republic). The CK were analysed byHPLC linked to a Quattro II mass spectrometer(Micromass, Manchester, UK) equipped with an elec-trospray interface [micro-LC-(ES + )-MS/MS]. Twenty- W ve microlitre of each puri W ed sample were injected ona C 18  reversed-phase Phenomenex column, purchasedfrom Bester BV (Amstelveen, The Netherlands).The Netherlands and eluted with a solvent gradient(MeOH and 0.01M NH 4 OAc) according to Prinsenetal. (1998). The chromatograms obtained were analy-sed by means of Masslynx software (Micromass) andthe CK concentrations were determined following theprinciple of isotope dilution. Results Isolation of mutants with short hypocotyl in the presence of ACC+GA 3 Ethylene and GAs are known to promote hypocotylelongation when  Arabidopsis  seedlings are grown in  834Planta (2007) 225:831–842  1 3 the light (Smalle etal. 1997; Cowling and Harberd1999). In addition, treatment with both ACC, the eth-ylene precursor and GA 3  was reported to have at leastan additive -and most often a synergistic- e V  ect onhypocotyl elongation, suggesting the existence of inter-actions between these two hormones (Saibo etal.2003). In order to identify factors that play a role in thisinteraction, we screened for mutants in which thehypocotyls do not elongate in response to the combina-tion of those hormones. Thus, 5,000 M2 mutagenisedseeds were grown for 9days on LNM supplementedwith 50   M ACC and 10   M GA 3 . Thirty-two 9-day-old seedlings were found with hypocotyls similar inlength to non-treated wild type seedlings. These seed-lings were grown on soil, selfed and the F1 progeny wasagain analysed on LNM+ACC+GA 3 . For 2(  ACC + GA  3  insensitive  seedlings, agi5  and agi16 ) of 32candidates a poor response to this hormonal treatmentwas con W rmed.Genetic mapping and determination of the agi16  mutationThe F1 progeny from the cross between agi16  and Col-0 had a wild-type phenotype (data not shown), indicat-ing that agi16  carries a recessive mutation. To identifythe genetic location of agi16 , 379 F2 seedlings srci-nated from the cross between agi16  and L. erecta  (Ler)were analysed, using the hookless phenotype as a char-acteristic trait of the agi16  mutation. Eighty-threeseedlings turned out to be homozygous for agi16 , thuscon W rming the recessive character of this mutation[segregation ratio wild type:hookless was 296:83,  2 =1.943, P  (  2 ¸ 1.323)=0.25]. Using SSLP markers,a  gi16  was mapped between F24M12-TGF and FUS6.2,at the bottom arm of chromosome 3. Given that dark-grown agi16  seedlings did not form an apical hook(hookless phenotype) even when treated with 10   MACC (Fig.1) and the recessive nature of the mutation,we reasoned that agi16  could be allelic to a knownhookless mutant.  Amp1 / cop2  are also located in thebottom arm of chromosome 3, thus indicating that agi16  might be another allele of these mutations.Therefore, we performed complementation tests with amp1-1  and hls1  (located in chromosome 4). The F1from reciprocal crosses between agi16  and hls1  gaverise to seedlings that form an apical hook, proving thatthese mutants carry mutations in di V  erent genes andtherefore can complement each other. In contrast, theF1 from reciprocal crosses between agi16  and amp1 showed a hookless phenotype, indicating allelismofboth mutants. Therefore, agi16  was renamed amp1-7  , as amp1-1 , amp1-2 , amp1-3  and three otheralleles were already reported (Mayer etal. 1991;Chaudhury etal. 1993; Hou etal. 1993; ChinAtkins etal. 1996; Mordhorst etal. 1998). Nucleotide sequence analysis of amp1-7   revealed the presence of a single point mutation (G–A at cDNA nucleotide1634; At3g54720), which is predicted to cause aglycine-to-glutamate substitution at residue 545 (G545 E)of the AMP1 protein. Figure2 shows that the amp1-7  mutation (four residues upstream of amp1-1 ) muta-tion is located in the C-terminal region of the AMP1peptidase domain. Fig.1 Cellular organization in the hypocotyl of dark-grown  Arabidopsis thaliana  seedlings. Four-day-old Col-0, amp1-1  and amp1-7   seedlings were grown on LNM supple-mented with 10   M 1-amino-cyclopropane-1-carboxylic acid (  ACC  ) and/or 10   M zea-tin in the dark  and analysed under di V  erential contrast ( DIC  ) microscopy. In order to clearly show the cell size in the di V  erent regions of the hypo-cotyl, some cells were outlined with a black line . Bar  =100   m  Planta (2007) 225:831–842835  1 3 Phenotypic analysis of amp1-7  The  Amp1-7   was isolated based on its altered responseto ACC+GA 3 . The triple response was used to furtheranalyse ethylene responsiveness of the mutant. In addi-tion to the absence of an apical hook in the presence of ACC, microscopic analysis showed that amp1-7   had adi V  erent cellular organization along the hypocotylwhen compared with wild type (Fig.1). Dark-grownCol-0 seedlings treated with ACC showed long cells atthe base and the midsection of the hypocotyl, followedby a region with intermediate cell size near the apexand small cells in the apical hook. In contrast, amp1-7  (and amp1-1 ) revealed the opposite pattern; small cellsat the hypocotyl base, intermediate sized cells in themiddle and the longest cells in the apical part of thehypocotyl. To investigate whether the di V  erent cellularreorganization observed in amp1  mutants was a conse-quence of the high-endogenous cytokinin levels weanalysed 4-day-old Col-0 seedlings grown in the dark inthe presence of either 10   M zeatin or 10   MACC+10   M zeatin. Both treatments induced apicalhook formation and the same cellular size distributionpattern as observed for Col-0 treated with ACC(Fig.1), indicating that the hookless phenotype as wellas the di V  erent cell organization in amp1  mutants werenot due to the general high-endogenous zeatin levels.The W rst striking feature of light grown amp1  seed-lings was the acceleration of leaf emergence comparedto Col-0 (Fig.3). In contrast to amp1-1  (20% were tri-cot or tetracot) (Chaudhury etal. 1993), no polycotylywas observed in amp1-7  . Fifteen-day-old amp1-7   seed-lings clearly revealed an intermediate phenotypebetween Col-0 and amp1-1  with respect to the leaf number. Furthermore, both 15-day-old amp1  mutantsshowed serrated leaves prior to full expansion. The lat-ter characteristic is typical for cytokinin over-produc-ers (Rupp etal. 1999). At 28days both amp1-7   anda mp1-1  showed a precocious X owering as comparedwith wild type (Fig.3).  Amp1-1   X owers 6–7days earlierthan wild type and 3–4days earlier than amp1-7  . Both amp1  alleles also revealed semi-sterility. A fraction of  X owers never formed siliques. Figure3 also shows thatadult mutant plants were shorter and more branchedthan wild type and had shorter siliques. It is also worthto mention that neither the semi-sterility nor theshorter siliques phenotype could be recovered by treat-ment (regular watering) with 10   M GA 3  or 50   MACC. Most of the amp1-7   features showed an interme-diated phenotype between Col-0 and amp1-1 , con W rm-ing that amp1-7   is the weaker allele.Hypocotyl elongation in amp1-7   is less responsive to ACC and GA 3 Figure4a displays hypocotyl length of Col-0, amp1-7  and amp1-1  seedlings treated with either 50   M ACCor 10   M GA 3 , or with the combination thereof. Hypo-cotyl elongation of amp1-7   seedlings was stimulated byeither ACC or GA 3  as was the case for Col-0, whereas,the elongation of amp1-1  hypocotyls was not signi W- cantly altered. When treated with the combination of ACC and GA 3 , Col-0 showed a 94% increase in hypo-cotyl length, whereas amp1-7   hypocotyls increased58% and amp1-1  only 38%. The weak response (ascompared to Col-0) of both amp1-7   and amp1-1 , led usto investigate the responsiveness of these mutants toeither ACC or GA 3  in more detail.First we determined the dose response curve of the amp1  mutant hypocotyls to ACC treatment both in thelight and in the dark. Hypocotyl in wild-type seedlingsis normally enhanced by ACC in the light, but inhib-ited by ACC in the dark. Figure4b shows that thehypocotyl of amp1-7   dark-grown seedlings was lessresponsive to ACC than Col-0. Treatment with 1   MACC caused a 60% inhibition in Col-0, whereas thesame e V  ect was only observed in amp1-7   at a ten timeshigher ACC concentration.  Amp1-1  appeared to beeven less responsive to ACC than amp1-7  , as 60% inhi-bition was not even reached on 10   M ACC. Whenseedlings were grown in the light in the presence of ACC (Fig.4c), amp1-7   also showed to be less respon-sive to ACC than Col-0. In Col-0, the hypocotyl lengthwas increased by 40% upon treatment with 1   M ACC,whereas the same relative increase in amp1-7   hypoco-tyls was only seen on 5  M ACC. Again, amp1-1 hypocotyls were the less responsive to ACC withenhancement of hypocotyl length never exceeding 10%.Figure4d shows that the hypocotyl elongationresponse in the presence of GA 3  was lower in bothmutants, amp1-1  and amp1-7  , as compared to Col-0.The hypocotyl length of Col-0 seedlings was enhanced Fig.2 Physical map of the AMP1 polypeptide. Grey boxes  indicatethe conserved domains: PA  protease associated,  peptidase_M28 peptidase family M28, TFR_dimer   transferrin receptor-like dimer-isation domain. Amino acid residues are numbered starting fromthe N-terminus.  Arrows  indicate the positions of mutations indi V  erent amp1  alleles. For each mutation, the nucleotide substitu-tion and consequent protein modi W cation are indicated
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