Welcome to Plant Developmental Biology course.So, in previous lectures we have finished some of the example of root development, today’sclass we will start with Shoot Development and particularly we will focus today on ShootApical Meristem Maintenance.So, if you recall some of our previous lecture.So, if we summarize here so, we have seen that during the development the basic bodyplan which is being established during the process of embryogenesis.So, if you look a typical mature embryo, here you can see that shoot and root system hasbeen already the body plan the basic body plan for root and shoot system is alreadyestablished.And one very important thing what happens that, the axis body axis is established andthen the root apical meristem and shoot apical meristems are positioned along the axis.And this is you have seen in root that this is very important for proper growth and development,particularly post embryonic development of different organs in both root system as wellas in shoot system.So, in this class we will focus now on the shoot system.So, shoot system after post embryonic development it makes mature plant, in mature plants youhave many part or many organ depending on the plant species.But if you look the basic structures so in basic structure we have stem, some branches,some buds then later on after transition from vegetative phase to reproductive phase, wehave some structure called inflorescence and in inflorescence we have flowers so, theseare the shoot systems part.And if you look the apex, very apex of the shoot which is called shoot apex, it has meristematicregion or the region where stem cells are basically positioned.Stem cells which is responsible for primary growth of the shoot and this is called shootapical meristem.So, we will see in details what is happening and how the shoot apical meristems are maintained,but important thing here is that at the shoot apex there are coordinated process is goingon.So, the coordination between stem cell maintenance as well as cell differentiation, two thingsare happening.And, some cells basically the cells which is in blue in color this is basically stemcell, and this is being maintained throughout the life or till the plant is growing, andthis is important.And when these cells enters in this other regions they when it leave its place or whenit leaves its domain it enters in the process of differentiation.And in the differentiation depending on the identity of the meristem it takes differentidentity or different phase.For example, if the meristem identity is shoot then it will make lateral organ for shootfor example, leaf, but if the meristem identity is inflorescence then this will undergo theprocess of floral development.So, this is a typical example you can see this is primordia P 0 P 1 P 2 different organsare developing and, but a still a balance between cell division and cell differentiationsare maintained.And this shoot basically after certain time point, it transit to the reproductive phase,now it makes flowers.In flowers it makes some fixed organs, sepals, petals, stamens and carpel and after makingcarpels in most of the flowers this process of stem cell maintenance is inhibited it istotally suppressed.So, that is why there is no longer or there is no longer maintenance of stem cells andthis is called determinate development.So, the indeterminacy get converted into determinate development.So, this is a view of shoot apical meristem and this is how shoot apical meristems areorganized.So, this is basically inflorescence meristem.So, if you look on the top, this is how Arabidopsis growing, Arabidopsis inflorescence looks theseare the different flowers, these are early flowers, these are mature flowers where youcan see that they have already come if you look the longitudinal view.So, this is how the meristem is maintained, this is the central meristem and then youhave the lateral organs coming.As I said depending on what is the nature of meristem if it is shoot meristem then thelateral organs will be leaf, when if it is inflorescence meristem the lateral organswill be flowers.Both are quite similar in process of establishment and differentiation.If you look the shoot apical meristem there are certain domains.So, the this domains which you look here this is called central zone, this is in the centreof the meristem then you have a layer just flanking the central region which is calledperipheral zone.And then you have a domain or zone which is below the central domain which is called ribzone and this is the region the green color is basically stem cell region.So, this domain is maintained and when the cells are present in the central zone or inthe stem cell niche, they will have a property of cell division.But once they leave the central domain and enters in the peripheral region or entersin the rib zone, they basically loses its stem cell property and then initiate differentiationspecific program.Another important thing the shoot apical meristem also has different layers.So, you can have this is the first layer which is called L 1, then the second layer whichis below the L 1 is called L 2 and then the layer beneath it is called L 3 layer.So, this is the organization of shoot apical meristem and this is again the schematic view.So, as I said that the in the central zone this region is your stem cells and this regionis also called organizing cells.This organizing cells is very very important.So, positioning of organizing center is very important.Because organizing center is equivalent to you can say it is not exactly same, but itis equivalent to QC of root apical meristem and it plays a very very important role instem cell niche positioning and maintenance.This is the scanning electron micrograph picture of the SAM you can see this is the centralregion and then these are the primordia or the lateral organs which are coming from thisshoot apical meristem.If you look the establishment when the meristem shoot apical meristem is established it happensat very very early stage of embryogenesis.So, if you recall previous class you will find that the first cell which start celldivision is zygote, and then zygote undergo the process of a series of cell division andit undergo different stages of embryo development.And during this stages at a particular stage if you look here this late state, this regionis basically assigned as a shoot apical meristem and this region as a root apical meristem.And then you have a kind of in mature seedling if you look, shoot apical meristem is positionedhere.This is picture if you look here, this is zygote, this is single cell zygote and thisis first cell embryo.So, first cell division has occur here you can clearly see two nucleus, and if you lookhere even the first cell division is asymmetric.So, in development asymmetric cell division particularly in plant development, asymmetriccell division is one very very important process and which is eventually giving rise to theproper developmental program or establishing proper developmental program.So, even in the zygote you can see here the first asymmetric cell division is basicallyestablishing the process of later development.And this process are basically regulated by the first asymmetric cell division is regulatedby two pathways.One path way which is mediated by YDA which is called YDA and another process is basicallyregulated by homeobox containing transcription factor WOX2, WOX8 and WOX9.And both of the pathway in the zygote they are basically regulating the process of asymmetriccell division.Interesting thing here is that this pathway is getting activated by SSP messenger RNAwhich is basically transmitted from the pollens.So, the first step of shoot apical meristem initiation or you can see even the first stateof embryogenesis begins with this activating these two pathways.And then later on they play very important role.So, there is a dynamic expression and their transcript expression domain of these geneswhich basically helps in establishing apical fate of the cells or basal fate of the cells.So, if you look here this is zygote.So, zygote basically express all three transcription factor WOX2, WOX 8 WOX 9.But after first asymmetric cell division WOX2 is restricted to the apical cell which issmaller in size whereas, WOX 8 and WOX 9 they retain their expression in the basal cell.Later on you can see at this stage 8 cell stage the apical most cells they retain WOX2expression whereas, the basal side of the apical cells they basically have WOX 9 expression.And, this is the region which retains both WOX 9 and WOX 8, and WOX 8 alone is basicallymaintained in this cell.So, this you can tell that that same transcription factors they are changing or their expressionpatterns are basically helping in definingin establishing proper apical and basal patterningduring the embryogenesis, and this is the schematic diagram you can see here.So, in basal fate after cell division at first cell embryo, the basal cell fate is basicallypromoted by WOX 8 and WOX 9.And then WOX 8 and WOX 9 somehow restrict WOX2 expression to the apical fate, and inWOX2 basically active PIN one basically which is a kind of regulator of auxin signalingpathway.And then this is another regulators and how they basically helps in positioning the stemcell or positioning or establishing shoot apical meristem during embryogenesis.MP is transcription factor which is AUXIN RESPONSE FACTOR 5.So, if you look here the messenger RNA of MP is expressed here in the apical side whereas,messenger RNA basically it overlap here with the TMO7 whereas, TMO7 protein is presenthere as well as here.And some other regulators of shoot apical meristem establishment and maintenance aregetting also activated during the process of embryogenesis.For example, you will see later on this WUSCHEL, CLAVATA 3, CUC, STM all these activators arebasically expressed or activated at particular stage in a particular cell.Some of them are very important which we will discuss in detail for example, this WUSCHEL-CLAVATAsignaling; WUSCHEL CLAVATA signaling is very very important and if you look the patternhere.So, if you look here WUSCHEL is getting activated and then CLAVATA is coming up.When CLAVATA is coming up, WUSCHEL is restricted here CLAVATA is on the upper layer.So, if you look at this stage of embryo.So, you can see that CLAVATA3 are somewhere here and WUSCHEL is post here how this ishappening we will see in the next slide.. And SAM establishment is basically regulatedin wox mutants, just now I said that WOX is just playing very important role, but if youhave just single wox2 mutant we do not have a very strong phenotype.So, then it; so, basically if you look the four mutants together wox 1, 2, 3 and 5 thenthe effect is quite significant and what you can see here?This is the expression pattern this is In-situ hybridization of CLAVATA 3 see.So, if you look at the very early stage of embryogenesis you can see that it is expressedhere at the tip then it basically remains here at the upper side of the shoot apicalmeristem.And here you can very clearly see with the fluorescent marker, how a different developmentalstages of the embryo.And then if you have wuschel mutant, when WUSCHEL is not present; at early stage ifyou see CLAVATA3 expression is ok, but later on the CLAVATA3 expression is disappearingok.And if you look this mutant in mutant basically what happens CLAVATA3 is not present evenfrom the start.So, CLAVATA3 which is very important regulator of shoot apical meristem is missing in woxmutant background.But when you put PHABULOSA ; PHABULOSA if you recall some of the previous class we havelittle bit discussed about it.This is a class of homeobox domain containing transcription factor and it is regulated bymicro RNA.So, if you put PHABULOSA this is basically resistance to the micro RNA.So, if you create some kind of point mutation at the side of micro RNA binding, then whathappens this gene no longer will regulated by micro RNA, then you can have only one regulation.And when you put back this PHABULOSA in wox mutant that what you can see that the expressionof clavata3 is coming up.So, this basically tells that they all this critical regulators; critical regulators ofstem cells they are basically playing role in shoot apical meristem.So, now if you look here what are the key regulators which are responsible for shootapical meristem maintenance?So, this is again just to recap you, this is the central zone, this is the rib zone,this is the peripheral zone L 1, L 2 layer and this is the lateral organs coming andif you look some of the mutants.So, this is basically wild type you can see shoot apical meristem very nice, but if youhave clavata mutant you can see that the shoot apical meristem or the meristem is slightlyenlarged.So, there is more or bigger meristem size.So, which suggest that, CLAVATA is playing some role in may be restricting shape andmay be size of the shoot apical meristem; wherever if you see wuschel mutant in wuschelbackground, you see meristem like structure, but it is it looks that it is terminated early.So, there is premature termination of the meristem here whereas here is the gain ofmeristem meristematic activity.But if you look this mutant this st¬m; shoot meristem less mutant here you do not see anymeristem, so which suggest that STM is working at very early stage to even initiate shootapical meristem specific program and if you do not have STM, the meristems are not gettingformed.But these genes they are more working slightly later stage, they are more important for maintainingthe meristem.So, if you look here the very general way what happens that, this CLAVATA3 is basicallyexpressed here and actually the expression of CLAVATA3 is activated by WUSCHEL.So, if you recall WUSCHEL is first expressed here.So, WUSCHEL is expressed early then wuschel is basically activating expression of CLAVATA3,but once it activate expression of CLAVATA3.Once CLAVATA3 is on CLAVATA3 is; so, basically CLAVATA3 is a kind of signaling peptide, itis received by receptor which is CLAVATA1 and once this signaling is activated it inhibitexpression domain of WUSCHEL.CLAVATA signaling pathway ensures that WUSCHEL expression domain or transcription of WUSCHELis restricted to domain of expression, and if you recall this looks quite similar tothe signaling modules, which we have during the root apical meristem development.There we have CLE40 is regulating the expression of WOX5 and this is quite similar.So, this is basically a negative feedback regulation.So, wuschel is activating CLAVATA3 and CLAVATA 3 is repressing wuschel domain.Another important regulator of embryos of the shoot apical meristem is auxin; same auxinplant hormone which is regulating almost everything.And, what happens that in shoot apical meristem?The balance between auxin and cytokinin is important and this balance is embryogenesisis regulated by WOX2 module.So, if you have WOX2 modules the balance between auxin and cytokinin is maintained, and thismaintained balance is basically required or it is very important for regulating or coordinatingor maintaining balance between cell division and cell differentiation.And if you have WOX mutants here the balance is basically disturbed and that is why yousee the defect.Another important thing in the apical domain so, in apical domains; this is apical domainin the peripheral of the apical domains, some of the genes are activated one of this geneis AHP6 and AHP6 basically is a negative regulator of cytokinin signaling.And if you look here it is very specifically expressed only in the peripheral domain ofthe apical regions and in WOX mutant this expression domain is disturbed and you haveAHP expression everywhere.Similarly, this is a kind of another marker which is known to express in the apical regionand in mutant background this is disappear.So, this suggest that WOX mutants are very very important in assigning a proper fateto the apical region of the meristem.So, this is if you look the apical region what is happening, in the apical side thereare peripheral domain and the central domain.So, in central domain WOX2 module is basically getting activated WOX2 module is regulating,homeodomain zip transcription factor through micro RNA micro RNA is also regulating wewill see may be in the later slides and in here in this domain.So, basically WOX2 is restricting expression of AHP6 and micro RNA in the peripheral domain.So, when you do not have a WOX2 that is why you can see that AHP6 expression is comingin the central domain.And this kind of pathway regulating a critical amount of auxin and cytokinin signaling pathwayand this critical balance is regulating stem cell initiation and a balance between stemcell division and cell differentiation program.Ok so, again this hormonal regulation is very important, it is extremely important for maintainingshoot apical meristem.If you look, this case; so, here is basically STM expression, STM express in the meristemthen CUC is a kind of transcription factor which regulates at the boundary of the organs.So, basically this is expressed here in the boundary between meristem and the organs thisis very important.And this boundary has to be maintained very clearly and then you have WUSCHEL, WUSCHELis expressed in the very center region of the meristem.If you look the responses of auxin cytokinin and gibberellic acid what you see that, cytokininis very dominantly present in the meristematic region in the stem cell region whereas, theresponse of cytokinin in organ primordia is very very low, but auxin has about oppositepattern.So, if you look the meristematic region; most of the meristematic region they do not havevery high auxin signaling whereas the primordia the organ primordia they have high amountof auxin signaling.Gibberellic acid is having quite similar to the auxin, but auxin is also having some responseshere.So, this suggest that maybe cytokinin is positively regulating or very important for regulatingthe meristematic activity in shoot apical meristem whereas, auxin is playing major rolein the process of organogenesis starting new organ or the peripheral or the lateral organinitiation and that is how it happen.So, if you look this structure this is central zone peripheral zone.So, in the peripheral zone if you have peripheral zone what is happening?You have high cytokinin auxin ratio.So, you have high cytokinin and low auxin.So, that is why and gibberellic acid is low and this pathway is basically what happeningthat and STM is present everywhere.So, STM is important, STM is a positive regulator of cytokinin signaling and negative regulatorof gibberellic acid signaling.So, what is happening STM is activating cytokinin signaling in the meristematic region, wheregibberellic acid is low auxin is low, then WUSCHEL is getting activated.If you have high amount of cytokinin, it activates ARR and then WUSCHEL also regulate.So, there is a kind of feedback regulation going on here.So, basically high amount of cytokinin and STM they ensures meristematic property, butif you come in this region where the organs are coming or the site where organogenesishas to start what is happening here, there is low cytokinin and auxin ratio.So, basically high auxin low cytokinin.If you have high auxin, auxin is basically inhibiting STM activity there it is inhibitingcytokinin activity and it is probably inhabiting CUC activity.And all this process is helping in initiation of organogenesis specific program and thisis together basically ensuring that in the meristematic region or in the central regioncell should be dividing, but in the region where primordia has to come their cell divisionhas to be restricted or the stem cell property has to be restricted, and should be more kindof organogenesis specific property this is another simple way of looking here.So, if you look the central region in central region STM is activating cytokinin biosyntheticenzyme which is IPT7 and then you have high amount of cytokinin and then cytokinin isregulating expression of cytokinin response regulators and then it is basically helpingin establishing WUSCHEL CLAVATA signaling or regulating WUSCHEL CLAVATA signaling pathway.On the other hand if you look YUCCA is auxin synthesis gene and then you have auxin, auxinis activating transcription factor auxin response factor, and they are basically inhibitinghere ARR activity.Loop is establishing the stem cell identity.But in the peripheral zone or in the region where you have to have a primordia basicallyMP is getting activated by high auxin and STM is getting repressed and this resultsin basically primordia formation in the peripheral zone.Apart from auxin and this key regulator of WUSCHEL and CLAVATA signaling pathway, microRNA has been identified as a very important regulator of stem cell property or shoot apicalmeristem function.For example, if you have dcl1 mutant, DCL1 is basically a system which helps in biogenesisof micro RNA or small RNAs.So, if you have mutant basically micro RNA pathways are getting disrupted, and if youlook here in the mutant background what happens that proper embryogenesis is not taking placeand if you look at this stage the double mutant has unorganized growth or development.There are different types of phenotype or different classes of phenotype, but eventuallywhat is happening that the proper organization and maintenance of shoot apical meristematicis not happening in this mutant background.And if you look the auxin response or the expression of STM because this proper auxinresponse has to be initiated.So, proper auxin maxima has to be achieved where it should be achieved if that is nothappening then organogenesis will not start.At the same time STM which is very important regulator of meristematic activity has tobe activated for maintaining.So, both the process has to be function properly in order to have a proper balance.And if you look in this mutant background neither proper auxin response are being generatedif you look here auxin response is very dis-localized or it is not at the right place.This is a kind of a typical auxin response in the wild type, but if you look in the dclmutants, they are very diffused kind of response it is not getting properly organized.At the same time if you look the dcl mutant and if you look the expression of STM promoteractivity in wild type, it is very nicely positioned and express in the right domain, but in mutantbackground the expression is no longer here and this is true for another marker for shootapical meristem.So, this suggest that when you have disrupted micro RNA pathway, essentially neither stemcell is maintained nor proper organogenesis is taking place because auxin maxima not beinggenerated or proper auxin responses are not getting generated here.So, this suggest that micro RNAs working as one of the critical pathway to regulate thesteps and activity in shoot apical meristem.So, if we summarize here; so, you can look here.So, this is a typical shoot apical meristem, you have stem cell activity going on herethen you have WUSCHEL expression domain CLAVATA3-WUSCHEL pathways are working here.And then WUSCHEL is negatively regulating here ARR7 and then this is a cytokinin signalingpathway and then LOG is basically cytokinin biosynthetic enzyme.So, STM is activating cytokinin biosynthesis through IPT7 and LOGs are getting activatedhere.So, eventually in this region we are having a verv high amount of cytokinin and the cytokininis basically maintaining along with the wuschel and ARRs it is maintaining here a proper stemcell property.But if you come in the in the lateral region or in the peripheral zone, where auxin ishigh and auxin is basically activating transcription factor this transcription factor MP is basicallyrestricting the domain of cytokinin domains here in the meristematic region, but hereit should be repressed so, that the organogenesis process should start.And if you look here, this is slightly complex slide, but just to generally tell you thathow micro RNA is basically helping in establishing a proper polarity or stem cell maintenance.So, here you can see the asymmetric distribution.So, if you look this side which is basically abaxial side and what happens that you havehigh amount of micro RNAs and that is why its target which is HD ZIP is low at thisside, and if you look towards the adaxial side, it is having high amount of HD ZIP andlow amount of micro RNA.So, even the gradient or the amount of micro RNA and a particular transcription factorwhich is regulated by micro RNA are very important in positioning the stem cell activity andorganogenesis together and maintaining a coordination between stem cell activity as well as organogenesis.So, we will stop here in next class we will continue shoot development part.Thank you very much.