Plant extracellular matrix metalloproteinases

Plant extracellular matrix metalloproteinases
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  Review  :Plant extracellular matrix metalloproteinases Barry S. Flinn A , B A The Institute for Advanced Learning and Research, Institute for Sustainable and Renewable Resources,150 Slayton Avenue, Danville, VA 24540, USA. B Departments of Horticulture and Forestry, Virginia Polytechnic Institute and State University,Blacksburg, VA 24061, USA.   Email: barry. 󿬂 inn@ialr.org Abstract.  The plant extracellular matrix (ECM) includes a variety of proteins with critical roles in the regulation of  plant growth, development, and responses to pests and pathogens. Several studies have shown that various ECM proteinsundergoproteolyticmodi 󿬁 cation.Inmammals,theextracellularmatrixmetalloproteinases(MMPs)areknownmodi 󿬁 ersof the ECM, implicated in tissue architecture changes and the release of biologically active and/or signalling molecules.Although plant MMPs have been identi 󿬁 ed, little is known about their activity and function. Plant MMPs show structuralsimilarity to mammalian MMPs, including the presence of an auto-regulatory cysteine switch domain and a zinc-bindingcatalyticdomain.PlantMMPsaredifferentiallyexpressedincellsandtissuesduringplantgrowthanddevelopment,aswellas in response to several biotic and abiotic stresses. The few gene expression and mutant analyses to date indicate their involvement in plant growth, morphogenesis, senescence and adaptation and response to stress. In order to gain afurther understanding of their function, an analysis and characterisation of MMP proteins, their activity and their substrates during plant growth and development are still required. This review describes plant MMP work to date, aswell as the variety of genomic and proteomic methodologies available to characterise plant MMP activity, function and  potential substrates. Additional keywords:  proteolysis, substrate, tissue inhibitor of metalloproteinase. Introduction The plant extracellular matrix (ECM) represents the region at thecell surface, outside of the cell membrane. In its simplest interpretation, the plant ECM is the cell wall, while a broader interpretation sees the ECM as the cell surface continuum,including both primary and secondary cell walls and theintercellular spaces of neighbouring cells. The mainconstitution of the plant ECM will re 󿬂 ect the polysaccharideand protein composition of the cell wall, but will also be are 󿬂 ection of cell type and location within the plant. For example, lipid molecules are considered ECM components of the cuticle-cell wall interface of epidermal cells ( Nawrath 2006),aswellascomponentsofthegynoeciumtransmittingtissueECM(Hristova  et al  . 2005). The plant ECM plays a signi 󿬁 cant role in plantgrowthanddevelopment.Inadditiontoservingastructuralfunction, the ECM helps in turgor regulation, serves as a protective barrier against pests and pathogens, and isintimately involved in cell-to-cell communication and signalling pathways (reviewed by Brownlee 2002; Humphrey et al  . 2007). Various aspects of growth and development ultimately involve modi 󿬁 cations to the ECM, including processes such as stigma development (Tao  et al  . 2006),embryogenesis (van Hengel  et al  . 2001), secondary cell walldevelopment(Goujon etal  .2003),pollentubegrowth(Jiang etal  .2005), root growth (Roudier   et al  . 2005) and the  Rhizobium  –  legume symbiosis (Brewin 2004).ProteinsrepresentaminorcomponentoftheECM,andmany,suchasglycine-richandhydroxyproline-richglycoproteins,playa structural role. In addition, there are several examples of extracellular proteins serving a signalling function. Theseinclude the cysteine-rich protein LeSTIG1 and its interacting partnerLePRK2,whichareinvolvedinpollentubegrowth(Tang et al  . 2004), small peptides (like the CLE family and PSK) and theirassociatedreceptors(reviewedbyFarrokhi etal  .2008),lipid transfer proteins (reviewed by Carvalho and Gomes 2007),arabinogalactan proteins (reviewed by Seifert and Roberts2007) membrane bound R proteins (reviewed by Martin  et al  .2003) and several plant growth regulator receptors (reviewed by Napier 2004). Protein degradation within the plant ECM The ECM protein composition contains intact secreted proteins,as well as proteins that have been subjected to proteolysis and/or  processing modi 󿬁 cation. The proteolytic processing of ECMmolecules has been well characterised in animal systems(Ye and Fortini 2000), and also occurs in plants. Plant ECM protein proteolysis occurs through the action of microbial pathogen-released proteases (Dow  et al  . 1998; Carlile  et al  .2000), as well as plant-produced extracellular proteases. Thetomato subtilase P69B was shown to process the extracellular tomato leucine-rich protein LRP during viroid pathogenesis(Tornero  et al  . 1996). In addition, the  Arabidopsis  extracellular  CSIRO PUBLISHING www.publish.csiro.au/journals/fpb  Functional Plant Biology ,  2008,  35 , 1183  –  1193  CSIRO 2008 10.1071/FP08182 1445-4408/08/121183  aspartic protease CDR1, when overexpressed, causedthe releaseof a small extracellular elicitor peptide leading to local and systemic pathogenesis-related gene expression (Suzuki  et al  .2004). Apart from these plant   –   pathogen associations, other ECM-localised proteases appear required for normal plant development, suggesting that the proteolytic modi 󿬁 cation of extracellular molecules is a necessity. Examples of theseinclude the extracellular serine proteases SDD1 (a regulator of stomatal distribution and density  –   Berger and Altmann 2000),ALE1 (a regulator of cuticle formation and epidermaldifferentiation  –   Tanaka  et al  . 2001), and the extracellular cysteine protease DEK1 (helps to maintain and restrict aleurone cell fate  –   Lid   et al  . 2002).An additional class of extracellular proteases, the matrixmetalloproteinases (MMPs), have been highly characterised inmammals, and play signi 󿬁 cant roles in their biological processes(Sternlicht and Werb 2001). Plant MMP-like proteins exist, but relatively few have been characterised, and little is known about theirrolesandmechanismsofactioninplantdevelopment.Giventheir importance in extracellular matrix remodelling and signalling in mammalian systems, the potential relevance of MMP structure, activity and function in plant systems warrantsfurther study. Matrix metalloproteinases: general structureand function as ECM modi 󿬁 ers The characteristics of matrix metalloproteinases have beendetermined primarily through a wealth of vertebrates studies,which have revealed that MMPs are members of the metzincin protease superfamily, characterised by a catalytic domain withthree histidine residues, which are involved in coordinating theactive site with zinc ion. This is followed by a conserved methionine (Met) turn, which sits below the active site zinc(Stamenkovic 2003). MMPs are calcium- and zinc-dependant,activeatneutralpH(VuandWerb2000),andhavebeenidenti 󿬁 ed in various vertebrates, invertebrates and plants (Massova  et al  .1998). Most research on MMP function has involved mammals,with 23 human MMPs identi 󿬁 ed to date (Page-McCaw  et al  .2007). MMPs were initially thought only to degrade structuralcomponents of the ECM, but it has become evident that throughtheir proteolytic activities, MMPs can modulate tissuearchitecture, allow cell migration, generate biologically-activemolecules through substrate cleavage, modify the activity of signalling molecules and generate new signalling epitopes bythereleaseofcrypticinformationfromdegradedECMmolecules(Page-McCaw  et al  . 2007). As a result of MMP activity, variousimportant cell physiological events are regulated, including theactivation of antimicrobial peptides, stimulation of apoptosis, boneresorption,facilitationoftumourinvasionandangiogenesis(Ii  et al  . 2006).In vertebrates, MMPs contain three conserved structuraldomains: an  N  -terminal auto-inhibitory propeptide, a catalyticdomain, and a hemopexin domain at the  C  -terminus, althoughminimal domain MMPs exist, lacking the hemopexin region(Massova  et al  . 1998). The propeptide is ~80  –  90 amino acidslong and contains a conserved sequence (PRCGXPD), known asthe cysteine switch. The cysteine residue within the conserved sequenceinteractswiththecatalyticzincatomtoinhibitcatalysis.The catalytic domain contains a calcium ion and two zinc ions,with a zinc ion at the active site, and a calcium ion. The zinc- binding motif of the catalytic domain contains the conserved sequence HEXGHXXGXXH, with the three histidine residuesresponsible for coordinating with the catalytic zinc ion. Thehemopexin-like domain, when present, plays a role in substrate binding (Overall  et al  . 2002) and possibly in interactions withthe tissue inhibitors of metalloproteinases (TIMPs), which areendogenous proteinaceous MMP inhibitors (Das  et al  . 2003).The majority of MMPs are secreted into the ECM, althoughthere are six membrane-type MT-MMPs that maintain contact with the cell surface, through either transmembrane domains or GPI-anchors ( Nagase  et al  . 2006). Most MMPs are secreted in alatentform,butasubsetareactivatedintracellularlyviatheactionof furin-like preprotein convertases (Stawowy  et al  . 2005), withfurin cleavage sites located between the cysteine switch and catalytic domain (Vu and Werb 2000). Activation of theinactive, secreted MMPs involves the sequential cleavage of the propeptide by proteolytic or non-proteolytic mechanisms(Chakraborti  et al  . 2003), disrupting coordination between thecysteine thiol in the propeptide and the zinc atom in the catalyticdomain. This cysteine switch mechanism can account for allmechanisms of MMP activation (van Wart and Birkedal-Hansen1990).ThecatalyticactivityoftheMMPcanfurtherberegulated  by interaction with members of the TIMPs, which bind to thecatalytic domain of the MMP and inhibit any catalytic activitythrough a complex formation between MMPs and TIMPs(Chakraborti  et al  . 2003). Plant MMP structure The important role of MMP action in extracellular matrixmodi 󿬁 cation and subsequent mammalian development and signalling suggests that an elaboration of plant MMP structureand function may reveal new aspects of ECM modi 󿬁 cationin plant development. The  󿬁 rst report of plant matrixmetalloproteinase activity was described as soybean ( Glycinemax  L.) Azocollase A by Ragster and Chrispeels (1979), whichwas subsequently puri 󿬁 ed and characterised as SMEP1 byGraham  et al  . (1991). MMP-like sequences have beenidenti 󿬁 ed in numerous plant EST collections (The Gene IndexProject   –   http://compbio.dfci.harvard.edu/tgi/, accessed 5January 2008), representing various tissues, organs and stressconditions. However, few of these have been characterised. Theemphasis on  Arabidopsis  sequence characterisation resulted in areportbyMaidment  etal  .(1999)whichdescribedthepresenceof  󿬁 veMMPs(At1-MMPtoAt5-MMP)inthe  Arabidopsis genome.A recent analysis of GenBank sequences for full-length (FL)openreadingframes(ORFs),usingtheconservedcysteineswitchand functional zinc-bindingmotifs from the  Arabidopsis  MMPs,revealed several proven proteolytically-active, as well as putative, functional plant MMPs (Table 1; Fig. 1), representing both expressed sequence tag and genomic DNAsequences.TheseanalyseshavealsoindicatedthatallplantMMPgenomic sequences are intronless (Pak   et al  . 1997; Maidment  et al  . 1999; Delorme  et al  . 2000; Liu  et al  . 2001; Combier   et al  .2007). All of these translated ORFs possessed signal peptidecleavage sites, in agreement with their role as secreted,extracellular proteins. All contained a characteristic cysteine 1184  Functional Plant Biology  B. S. Flinn  switch PRCGXXD motif, with the exception of the soybeanGmMMP2 sequence, with an LRCGVPD sequence instead. Inaddition, they contained the functional catalytic domain zinc- binding motif (HEIGHXLGLXH), followed by the conserved methionine residue of the Met turn. Interestingly, several variant legume sequences have also been identi 󿬁 ed with an E to Qsubstitution in the zinc-binding motif of the catalytic domain(Combier  etal  .2007),andthesearepresentatahigherfrequencythan the non-variant sequences. This E residue is essential for  protease activity, and the E to Q mutation dramatically inhibited mammalian MMP proteolytic activity (Rowsell  et al  . 2002).Hence, the representation of these legume MMP-likemolecules as functional extracellular proteases remains inquestion. A relatively conserved, plant unique sequence (DLE/ QS/TV/A) of unknown function was also present on the  N  -terminal side of the zinc binding motif (Maidment   et al  . 1999;Fig. 1). Several of the FL MMPs also contained at least one putative  C  -terminal, GPI-anchor modi 󿬁 cation site and/or a  C  -terminal transmembrane domain (Table 1; Clark   et al  . 2004),suggesting potential extracellular membrane attachment.However, there has been no con 󿬁 rmation of membranelocalisation for any plant MMP to date. Furthermore, some of the MMPs contained putative furin cleavage sites(Table 1), located between the cysteine switch motif and catalytic domain, within 46 amino acid residues of thecysteine switch motif. It is unknown whether these predicted cleavage sites are real, and if so, if they are of functionalrelevance. Phylogenetic tree analyses of predicted plant MMPsequencesfrom38translatedplantMMPsequencesrepresentingtheregionspanningthecysteineswitchtoMetturn,indicatedthat the legume variants described above, and GmMMP2 are aseparate group compared with other plant MMPs (Combier  et al  . 2007), distinguished by the absence of the structuralcalcium and zinc binding sites. Overall, the plant MMPsequences resembled the minimal MMPs (mammalian MMP-7and MMP-26) in that they lacked a  C  -terminal hemopexin-domain. Although several plant MMPs have been described,there has been some confusion regarding the naming conventionfor these enzymes. Clark   et al  . (2004) suggested a system based on the  Arabidopsis  MMPs they characterised (Maidment   et al  .1999), to avoid confusion and assumption of similarity tovertebrate MMPs. Con 󿬁 rmation of plant MMP activity All plant MMP classi 󿬁 cations are based on sequence motif and structure conservation. However, an understanding of MMPinvolvement in plant development is dependent on the Table 1. Representative putative full-length functional MMP ORFs identi 󿬁 ed in GenBank NR  Thecysteineswitchandzinc-bindingmotifsof   Arabidopsis MMPswereusedforaBLASTsearchagainsttheGenBankNRdatabase.TheORFswereidenti 󿬁 ed andcysteineswitchandcatalyticdomainzinc-bindingmotifsdetermined.Theywerealsoanalysedwithavarietyofpredictivesoftwareprogramsforsignalpeptidecleavagesite(SignalP3.0:http://www.cbs.d.tu.dk/services/SignalP/,accessed14January2008),furincleavagesiteprediction(ProP1.0:http://www.cbs.d.tu.dk/ services/ProP/,accessed14January2008),GPIanchorprediction(BIG-PIPlantPredictor:http://mendel.imp.ac.at/sat/gpi/plant_server.html,accessed14January2008)and  C  -terminaltransmembranedomainprediction(LocalizomeTransmembraneTopologyPredictor:http://localodom.kobic.re.kr/LocaloDom/index.htm,accessed 14 January 2008). The GPI-anchor prediction includes both primary and secondary prediction sites when present Plant species Gene name/ identi 󿬁 er GenBank Accessionno.Lengthin aminoacidsSignal peptidecleavagesite predictionCysteineswitchmotif Furincleavagesite predictionCatalyticdomainzinc-bindingmotif GPI-anchor  prediction C  -terminalTM prediction  Arabidopsisthaliana At1-MMP (At4g16640) NM_117765 364 RFG-AR PRCGVSD No HEIGHLLGLGH No YesAt2-MMP (At1g70170) NM_105685 378 ASA-WF PRCGNPD NRR-DL HEIGHLLGLGH WRIDG YesAt3-MMP (At1g24140) NM_102260 384 VSA-GF PRCGNPD NRR-DL HEIGHLLGLGH No YesAt4-MMP (At2g45040) NM_130068 342 IEA-RN PRCGFPD WTR-DV HEIGHVLGLGH TNLAD NoAt5-MMP (At1g59970) NM_104689 360 ISA-KF PRCGNPD No HEIGHLLGLGH QSTGG Yes Glycine max  SMEP1 U63725 305 VSA-HG PRCGVPD No HEIGHLLGLGH No NoGmMMP2 AY057902 357 SDG-VS LRCGVPD No HEIGHLLGLDH NVEDS Yes Cucumis sativus  Cs1-MMP AJ133371 320 NTS-SP PRCGVQD No HEIGHILGLQH No No Vitis vinifera  N/A AM427487 354 IIP-DY PRCGVSD No HEIGHILGLAH No Yes N/A AM473484 353 CQP-GR PRCGMRD No HEIGHLLGLAH No No N/A AM442180 373 VSA-RF PRCGNAD No HEIGHLLGLGH TTNDS No N/A AM453535 319 ANG-EN PRCGVAD No HEIGHLLGLGH No No N/A AM449782 303 ATS-s.d. PRCGVPD No HEIGHLLGLAH No No  Medicagotruncatula MtrDRAFT_AC144345 g5 AC144345 368 VSA-RF PRCGVAD No HEIGHLLGLGH DRDSS YesMtrDRAFT_AC146588 g23 AC146588 373 VSA-RL PRCGVAD MKK-VV HEIGHLLGLGH NIGNG No Oryza sativa  Os02g0740700 NM_001054610 372 AMA-FP PRCGVAD No HEIGHLLGLGH EMDGS YesOSJNBa0010C11.5 AC069300 355 VHG-HG PRCGVGD No HEIGHVLGLGH TSSSS NoOs06g0239100 NM_001063794 371 AFA-LP PRCGVAD No HEIGHILGLGH MDSAG No  Mimulus gutattus  N/A AC182562 374 ASA-NF PRCGNAD No HEIGHLLGLGH RDTSG No  Lotus japonicus  N/A AP006395 382 VVS-AR PRCGVAD TKK-VV HEIGHLLGLGH PERDA Yes Plant matrix metalloproteinases  Functional Plant Biology  1185  con 󿬁 rmation of MMP activity, since their classi 󿬁 cation assumessome functional relevance based on proteolytic activity. Severalassays exist for the detection of MMP activity (Lombard   et al  .2005; Snoek-van Beurden and Von den Hoff 2005). All of the assays have been developed for the detection of mammalianMMPs, and utilise actual protein substrates, synthetic peptidesand synthetic mini-collagens.Plant MMP activity has been assessed using the degradationof general protease substrates like myelin basic protein (MBP)and Azocoll. Using one, or both of these substrates, recombinant At1-MMP (Maidment   et al  . 1999), recombinant soybeanGmMMP2 (Liu  et al  . 2001) and puri 󿬁 ed soybean AzocollaseA/SMEP1 (Ragster and Chrispeels 1979; Graham  et al  . 1991)were con 󿬁 rmed as possessing protease activity. Quenched  󿬂 uorescent test peptides, based on short amino acid sequencescontaining the scissile Gly-Leu/Ile bond of collagen, have also beenusedtocon 󿬁 rmtheproteaseactivityofcucumberCs1-MMP(Delorme  et al  . 2000), soybean SMEP1 (McGeehan  et al  . 1992)and At1-MMP (Maidment   et al  . 1999).Zymographic studies using either gel-embedded casein or gelatin have been used to demonstrate protease activity of various mammalian MMPs, although differential capacities for gelatin and casein degradation have been demonstrated,depending on the MMP involved (Yu and Woessner 2001).Zymographic methods have provided mixed results with plant MMPs,suchthatalthoughzymographyhascon 󿬁 rmedtheactivityofCs1-MMP(Delorme etal  .2000),itdidnotworkforAt1-MMP(Maidment   et al  . 1999). Modi 󿬁 cations to casein or gelatinzymographic techniques by the addition of heparinsigni 󿬁 cantly enhanced the detection of proteolytic activity of several mammalian MMPs (Yu and Woessner 2001). Thismodi 󿬁 ed procedure has yet to be tested with plant MMPs, but might enhance the gel-based zymographic detection of plant MMPs.The use of metalloproteinase inhibitors has also helped toverify the nature of plant MMP activity. Chelators (includingEDTA,EGTAand1,10-phenanthroline),aswellashydroxamatecompounds (Batimastat or BB-94), which act as general ( A )( B  )Cysteine switchmotifZinc-binding motifand met turn *GPI-anchor GPI-anchor modification site odification site *Furin cleavage site Furin cleavage site *Transmembranedomain Fig.1.  Plantmatrixmetalloproteinases(MMPs).(  A )GeneralstructureofplantMMPs,withrelevantdomainsidenti 󿬁 edandcolour-coded.Positionsofputativefurincleavagesites,GPI-anchormodi 󿬁 cationsitesand  C  -terminaltransmembranedomains,whenpredicted,areindicated.(  B )Alignmentofthecysteineswitchandzinc-binding/Met-turnmotifsfortheplantMMPslistedinTable1.SequencealignmentsandimagingweregeneratedusingPRALINE(SimossisandHeringa 2005; http://zeus.cs.vu.nl/programs/pralinewww/, accessed 14 January 2008). 1186  Functional Plant Biology  B. S. Flinn  mammalian MMP inhibitors, also inhibit plant MMP activity(Graham  et al  . 1991; Maidment   et al  . 1999;Delorme  et al  . 2000;Liu  et al  . 2001). In addition, plant MMP activity is inhibited bysome mammalian TIMPs. Applications of TIMP-1 and TIMP-2inhibited the activity of At1-MMP (Maidment   et al  . 1999),TIMP-1 inhibited SMEP1 activity (McGeehan  et al  . 1992),and Cs1-MMP activity was slightly inhibited by TIMP-1, but not TIMP-2, -3 or -4 (Delorme  et al  . 2000).MMP activity assays have used both puri 󿬁 ed native and recombinant proteins. The puri 󿬁 ed native protein studies haveusedsoybeanAzocollaseA/SMEP1,andotherplantstudieshaveused various forms of recombinant proteins for their activitycharacterisation. Recombinant proteins lacking the prodomaincysteine switch motif were signi 󿬁 cantly more active proteolytically than the pro-MMPs (Maidment   et al  . 1999; Liu et al  . 2001). In some cases, the recombinant proteins weresynthesised without the prodomain (Delorme  et al  . 2000; Liu et al  . 2001). In the case of At1-MMP, the MMP activator 4-aminophenyl mercuric acetate (APMA) was used to cleavethepropeptidedomainandactivatetheprotease(Maidment  etal  .1999), although MMP self-processing in the absence of APMAhas also been observed (Delorme  et al  . 2000). Plant MMP expression Publishedreports onplantMMPgeneexpressioncoveralimited numberofplants(  Arabidopsisthaliana (L.)Heynh, Glycinemax (L.) Merrill,  Cucumis sativus  L.,  Medicago truncatula  Gaertn.)and a limited number of growth/development/stress conditions.Several reports have indicated an increase in MMP transcript levels in association with aging and/or senescence. Early studieson soybean SMEP1 (Pak   et al  . 1997) indicated an increase intranscripts in 10-day-old leaves, with little subsequent change inlevelby30days,andnoaccumulationinrootsorstems.Inconcert withtranscriptabundance,SMEP1proteinaccumulationwasalsodetectedinleavesofa21-day-oldplant,primarilyin1st,2ndand 3rdtrifoliateleaves,representingleavesgreaterthan10daysold.The other reported soybean MMP, GmMMP2, also displayed detectabletranscriptlevelsinmatureleaves,withdeclininglevelsduring senescence (Liu  et al  . 2001). In addition, Delorme  et al  .(2000) conducted differential screening for genes expressed during senescence and the early onset of programmed celldeath (PCD) and identi 󿬁 ed Cs1-MMP as a gene which wasexpressed at the developmental boundary of senescence and PCD in cucumber, as well as in senescing male  󿬂 owers.In addition to the above patterns of gene expression,modulation of MMP transcript levels are associated with bothabiotic and biotic inputs. Soybean GmMMP2 transcriptsaccumulated in leaves in response to dehydration and wounding stresses, although the application of the stress-associated signalling molecules salicylic acid and jasmonicacid did not stimulate transcript accumulation (Liu  et al  .2001). At the level of biotic interactions, GmMMP2 transcriptsincreased in leaves during incompatible and compatibleinteractions following pathogen inoculation, as well as in cellsuspensions following elicitor treatment (Liu  et al  . 2001). Plant   –  symbiont interactions have also been shown to involve MMPtranscript level changes. As mentioned earlier, several variant MMP-like legume sequences have been identi 󿬁 ed containing anE to Q substitution in the catalytic zinc-binding motif. Theexpression of one of these, MtMMPL1, was studied in  M. truncatula  (Combier   et al  . 2007). Expression was onlydetected in young, developing root nodules, and not in shoots,stems 󿬂 owersorseedpods.MtMMPL1transcriptsweredetected  by3dayspost-inoculationwith Sinorhizobiummeliloti ,butnotinsymbiotic interactions between  M. truncatula  and arbuscular mycorrhizal fungi. In addition, the expression of MtMMPL1was localised in inner noduletissues within developing infectionthreads, and was triggered at the onset of infection thread formation.ThemostcomprehensiveplantMMPgenefamilystudieshave been conducted with  A. thaliana  (Maidment   et al  . 1999), whichhas  󿬁 ve MMPs (At1-MMP to At5-MMP). All  󿬁 ve At-MMPsexhibiteddifferentialexpressionpatternsin 󿬂 ower,root,stemand leafsamples(Maidment  etal  .1999),basedonRT  –  PCRanalyses.At1-MMP displayed strong levels of expression in  󿬂 ower, root and stem. At2-MMP exhibited strongest expression in roots, and At3-MMP was strongest in leaf and root, with expression in leaf vascular bundles. The expression of At3-MMP was alsodevelopmentally regulated and increased in  󿬂 ower and leaf at later stages of development. At-4 MMP displayed strongest expression in stem, and was also present in imbibed and strati 󿬁 ed seed, compared with dry seed. In addition, At5-MMPhadsimilar,strongexpressionlevelsinleaf,rootandstem.AmorecomprehensiveanalysisofAt2-MMPexpression(Golldack  etal  .2002) was conducted with young (4-week-old) rosette stage plants and older (10-week-old)  󿬂 owering plants. In young plants, At2-MMP transcripts were present in leaves and roots,with higher levels in leaves. In contrast, in  󿬂 owering plants,transcripts were detected in roots, leaves and in 󿬂 orescences, but  predominantly in the roots. Transcript abundance was alsoassociated with plant maturation in leaves and roots. Therealso appeared to be a maturation-associated, tissue-speci 󿬁 ccompetence for response to stress conditions or signallingmolecules. In young plants, methyl jasmonate (MeJA) and cadmium stimulated At2-MMP transcript accumulation inleaves, but sodium chloride (NaCl) did not. However, NaClstimulated transcript accumulation in roots, but MeJA and cadmium did not. In contrast, the roots of   󿬂 owering plantsshowed no transcript accumulation in response to any of thestresses, leaves and in 󿬂 orescences showed no transcript accumulation from MeJA, and a reduction of transcript accumulation in response to cadmium. At the cellular level, in situ  hybridisations with  󿬂 owers revealed At2-MMPexpression in mesophyll cell layers of the receptacle, in thecell layers near the epidermis of the gynoecium, and in theovules. In leaves, transcripts were present in vascular tissue(phloem and protoxylem cells), in addition to epidermal and mesophyll cells (Golldack   et al  . 2002).The relatively few studies described above have provided anincomplete view of MMP gene expression during development and response to stress. The availability of   Arabidopsis  genomicsresources and tools, such Genevestigator (https://www.genevestigator.ethz.ch/, accessed 10 January 2008;Zimmermann  et al  . 2004) and the Botany Array Resource(http://bbc.botany.utoronto.ca/, accessed 10 January 2008; Plant matrix metalloproteinases  Functional Plant Biology  1187
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