Welcome back to the course of Plant Developmental Biology. In today's class we are going todiscuss about Root Development. So, as we discussed in some of the previous class thatif you take a mature plants, this can be divided into two system, one is the root system andshoot system. Root system is usually below the ground part of the plant; shoot systemis above the ground part of the plant. And if you look a closer view of the verytip of the plant, at shoot this is a scanning electron micrograph picture of growing shootapical meristem which is basically inflorescence meristem. And at the very tip of the root,this structure is called root apical meristem which we will look in detail.So, this slide also you have seen in one of the previous class. So, if we take a generalroot development in plant, so there are three major developmental processes which is goingon during the development. First process which is called root apical meristem maintenancewhich occurs at the very tip of the growing primary roots, this is a typical primary rootsin model dicot plant. And if you look at the very tip this is theregion which is called shoot root apical meristem, and this is the region where some cells retaintheir identity as a stem cells. Stem cells means they can only undergo the process ofcell division, they do not enter in the process of cell differentiation.And this is very important for continuous growth of the root, for development of theroot, for growth of the root or for lot of organogenesis, always a continuous productionof cells are required. And this is happening here in the root apical meristem. And thenif you come slightly later zone of or slightly upper zone of the root, this zone is calledzone of cell expansion. And during this zone mostly tissue patterning the second part ofthe development in the root takes place, where different tissues or different layers of thecells they take a very special identity. And then if you come in the even higher upregion of the root the process of proper differentiation of new organs or organogenesis occurs whichis mostly in case of root is lateral roots or root branching. So, the process of rootbranching is visually initiated in the differentiation zone and a lot of lateral roots are developedin this region.So, now in in this class, we will focus on the first aspect and which is root apicalmeristem. What happens in the root apical meristem, what are the process, how the meristemis maintained, this is more important. Because if you look the root tip carefully, so, inthis picture, you can see this is the region which is basically the meristematic region;meristematic region is the region which undergo the process of cell division mostly. And thisis the region of cell elongation and then here is the differentiation.And if you make a closer view, this is how root tips looks. And if you make a cross sections,so you can clearly see that these are the different cell layers, the outermost layeris epidermis, then you have a layer of cortex, then you have endodermis, then this is pericycle.In pericycle there is a xylem axis. So, this is protoxylem, this is metaxylem.And then at two poles you have phloem poles, where you have sieve elements as well as companioncells and then you have some procambium cells. This is a longitudinal view. So, you can seethis is epidermis, cortex, endodermis pericycle and the vascular tissues. But apart from thatyou have some tissues here which is called columella cells and these are lateral rootcap. So, all these are part of a particular growing roots.How this process occurs? So, the first thing is that how the apical meristem is established,how this organization or proper organization of the root meristem takes place, and whatis the importance of all these cells? And then this happens at a very, very early stageof the embryogenesis during the embryogenesis. So, if you again recall one of our previousclass, where we have discussed that the first cells, first diploid cells after fertilizationis zygote. Zygote undergo the process of embryogenesis where it undergo several round of mitoticcell division. And at some stage for example, if you look at late globular stage, you cansee there are three domains, three distinct domains. This domain is eventually the upperdomain or apical domain which is eventually going to make the upper part of the plant.Then you have a middle domain here, this will make eventually the hypocotyl region of theseedling. And this lower domain is basically going to make the root.So, important thing here is that the program the basic developmental program for root,shoot is established at the stage of embryogenesis itself. Here you can clearly see that thisis the region of the root apical meristem, which is already established. Now, if youlook the growing root once it is emerged and you can see this tip this is the very closerand high magnification view. So, few cells which you have to understand very early tounderstand the entire process of root development. So, if you look this is the root tip, andthis red colour cell this is very important cell. So, you can see here tw o, but actuallythey are four in numbers. These cells are called quiscent centre, this is QC. And thefeature of these cells are that they themselves are very slowly dividing or almost not dividing,but they regulates the division capability of the cells which are close to them.So, if you look this boundary domain, this is the region which is called stem cell nichehere, because the cells which are lying in these domains they will not undergo the processof cell differentiation, they will only divide, they are the stem cells. These are also calledin plant initial cells. And one important thing here is that and youwill also realize later on that all these initial cells or the stem cells, they aredirectly connected with the QC which means that the cells which are directly connectedwith the QC they might be receiving some kind of signals which basically allow these cellsto remain as a stem cells or the cells which are dividing and it also helps them not undergoingthe process of differentiation. Then apart from that you can see these cellsdepending on their position this initial these are like stele initials. And if you look herethis is initial and actually for cortex and endodermis you have a common initial whichis called C E I, which is Cortex Endodermis Initial. But for epidermis and these initialsare basically if you look this the D 1 layer which is just below the QC these are initialfor columella cells. So, this entire region is columella.In columella, this immediate down to the QC, they are the intial, they are the stem cellscolumella stem cells. But other cells which are in D 2 layer, D 3 layer and D 4 layer,they have undergone the process of differentiation; columella specific differentiation. And thesecells are basically making lateral root caps. Another important thing which is importanthere to know that this columella cells when they undergo the process of cell differentiation,they accumulate starch and there are way to detect the starch.So, if you detect the starch, if you see the starch accumulation in the cell which is amarker that this cells has undergone the process of differentiation. These cells are no longerworking as a stem cells. And this is important. And then these different layers you can seeclearly epidermis layer, cortex layer and endodermis layer and stele layers they arearranged in a radial manner.So, next the thing was that there was report when it was studied that what is the importanceof QC because QC looks like that it is sitting in a very central position and it is regulatingstem cell property. And what is actually it is importance, how it is doing this function?And one very specific experiment was done, if you again recall in in one of the classwe have discussed about the experiment which is called laser ablation which through whichyou can specifically kill a particular cell in the in the growing root. And what happensif you do if you kill the QC? So, for example, this is a typical same structurewhich you have seen in the previous slide. So, if you look this picture, so this is yourQC, one of the QC is here, but second QC which is now very small here this QC is ablated.And if you ablate the QC and look what happens to the cells which are in contact with thisQC. You see here this is starch granules accumulation just now I say. So, if you see this starchgranule accumulation which means that these cells are not stem cells, but these cellsare undergoing the differentiation. But if you look this is the QC and just belowthe QC the cells which is in D 1 layer, they do not accumulate the starch which means thatthey are they are basically stem cells, but the cell layers below the D 1 like D 2, D3, D 4, they start accumulating the starch which means that they have lost the stem cellproperty and they have started cell differentiation. But if you look this case where one of theQC was depleted, the QC which is intact the layer which is below the QC or the cell whichis below the QC in the D 1 layer or which is called columella stem cells. This is stillmaintaining the stem cell identity, it has not accumulated starch. But the cell whichis below the ablated QC, this has started accumulating starch which means that thiscell has lost its capability of stem cells and it has started differentiation, so whichtells that QC is very very important in maintaining a cell as a stem cell identity.How this is possible, what could be the hypothesis? In first hypothesis, what can happen thatQC is activating division and repressing differentiation simultaneously, independently. And secondpossibility is that QC is actually activating, division and the result is inhibition of differentiation.In third possibility, QC is actually repressing differentiation and result is activation ofcell division. But if you look this experiment of you ifyou think this data, this suggests that the third pathway is working. Then on the basisof this, one model has been proposed. And the model here is that there are basicallytwo types of signals. One signals which might be coming from the very mature cells of theupper region of the root, and they are the differentiation signal they are promotingthe differentiation. And another signal which is coming from the QC going out from the QC,and this signal is basically inhibition of differentiation.So, basically both the balance between these two signals are might be important. The cellsand this signal is more kind of short distance signal. So, the cells which are in directcontact with the QC they are receiving this differentiation inhibition signals, whereasthese signals are coming from top. And what happens? This and here if you have inhibitionsignal, there the differentiation is inhibited here.But once this cells divide, so let us assume if this is an initial cell and if this celldivide, the cell which is present at the upper side, now it loses the contact with the QCwhich means that it might not be getting the signal the inhibition signal or the signalwhich is responsible for differentiation inhibition. But at the same time it is getting signal;it is getting signal from the top which is to promote the differentiation. Now, thesecells will start the process of differentiation, whereas the lower cell which still remainsin contact with the QC, it will retain the process of cell division and process of celldifferentiation is inhibited in these cells.So, there are the next question comes that ok, QC is important for maintaining cell differentiationa balance between cell division and cell differentiation, this is crucial. What could be the geneticpathway, what are the regulators, and what are the regulators, and how they helps inpositioning these stem cell niche in the root apical meristem? And one of the pathway isSCARECROW SHORTROOT protein mediated pathway. So, SCARECROW and SHORT ROOT, these are twoGRAS family of transcription factor, they are a special transcription factor and theyare expressed in a tissue specific manner. So, here if you look the expression patternof SCARECROW promoter activity of SCARECROW driving GFP expression, so it expressed verywell in the QC and the initials for cortex and endodermis. But at the later stage theexpression is only restricted to the endodermis, and there is no expression in the cortex.But in scarecrow mutant what happens that the expression in QC disappears, this region.Similarly, if you look other marker these are the QC 25 and QC 46, they are the markergenes which are known to express in the QC. But in scarecrow mutant background you cansee that the expression is totally lost, which means that in the scarecrow mutant QC identityis defective, which tells that the identity of QC is regulated by SCARECROW. But thisis not always some of the QC markers they still get expressed in the QC. So, may bethe identity is not completely lost or maybe this could be the position dependent expressionof the genes. But another interesting thing if you lookhere, so this is wild if you look here the granules accumulation starch granule accumulation,so if you look in the wild type this is the QC and this is the columella stem cells thereis no granule accumulation. But in absence of SCARECROW protein, the cells just belowthe QC they start accumulating starch. So, this tells that the stem cell maintenancecolumella stem cell maintenance is defective in case of scarecrow, this is this you canalso be seen here. So, this Q 1630 is marker for differentiatedcolumella cells. So, as you can see in the wild type case, this is QC and this layer,the D 1 layer which has columella stem cells. This gene is not expressed here, but the D2 layers which has the differentiated columella cells express Q 1630. But in scarecrow mutantbackground, you can see that the cells which is just below the QC can start accumulatingcolumella specific markers. This all together suggest that SCARECROW is important in notonly taking QC as a proper identity, but also positioning stem cell initial at the rightposition.Another important pathway or genetic regulation of positioning root stem cell niche is mediatedby another class of transcription factor which is PLETHORA. PLETHORA’S are AP 2 domaincontaining plant specific transcription factor. And this is very very important and if yourecall one of the class we have discussed that usually they work in a genetically redundantmanner, and that you can see here as well. So, when you have a single mutants, you donot see a very strong phenotype, but when you combine plethora1 and plethora2 doublemutant, you can see that root growth is inhibited. So, it is a very short root plant.And if you look the differentiation pattern, so this is the QC again the same thing. Butif you look the double mutant the cells just below the QC in the D 1 layer, it has startedaccumulating starch, which suggest that in double mutant plethora1 and 2 double mutantsthe stem cell niche is not getting maintained stem cells are losing their stem cell property.And thus this is also visible from here. If you look here this is double mutant plethora1and 2 double mutant, this is wild type. And this is the basically size of meristem. So,in wild type, this is the meristem size and this meristem size is significantly reducedin the double mutant. Here if you look the marker, cyclin gus marker,cyclin markers are basically markers which marks the dividing cells, it will expressin the cells which is under process of cell division. And if you look in the wild type,you can clearly see that large number of cells are under the process of cell division, butin double mutant this number is significantly reduced. Same thing you can look by lack ofthe QC 25 markers. So, in wild type, you have QC 25 markers expressed in the QC, but inmutant this the expression is totally disappeared. But then the question is that we know thatthere is already regulator SCARECROW and SHORTROOT pathways regulating positioning of the rootstem cells. Now, we are coming that PLETHORA’S are also regulating. Do are they working throughthe same pathway or they are working independently? So, to check that if you look these mutants,so basically this is the double mutant. And if you look the expression pattern of SHORTROOT,SCARECROW protein and SHORTROOT protein, and what you find that the expression looks quitenormal. So, in the embryo stage between wild type and mutant here also wild type and mutant,so the expression pattern of both SHORTROOT and SCARECROW protein is not significantlyaffected in plethora mutants. This suggests that they might be working independently forregulating a stem cell niche positioning in case of root apical meristem ok.So, if we combine here, so what you can see here that there are two pathways SHORTROOT-SCARECROWpathway and PLETHORA’S pathway. SHORTROOT SCARECROW pathway basically they are helpingin positioning QC in radial manner.Whereas so radial manner is basically in this manner. So, if you look this is the QC, sothey are expressed here SHORTROOT proteins are. So, if you look this picture this isthe expression pattern of the genes. So, this MP we will talk maybe later on this is a kindof genes which is regulated by auxin. And auxin is very important hormone which playsa very important role in the root development. But apart from that if you look the PLETHORAexpression, PLETHORA’S are expressed here in this domain. But at the later stage, ifyou look or maybe in the growing meristem if you look so this is the cells this is thecells where PLETHORA are expressed, this is the cell where PLETHORA are expressed. Thisis the cell where your SCARECROW and SHORTROOT proteins are expressed.But if you look this cell, so this is basically QC as well as this CEI initials, so cortexendodermis initials. These are the cells where all three genes are expressed PLETHORA, SCARECROWand SHORTROOT. So, basically the hypothesis is that SHORTROOT and SCARECROW PLETHORASare helping in positioning the stem cell niche in the radial manner, whereas PLETHORA’Smight be helping in positioning the stem cell niche in the longitudinal manner. And thisis this is very important. So, later on we will see that actually SHORTROOT activateexpression of SCARECROW, and it activates the expression of WOX 5 which we will seein another slides.So, here are there are two pathways which are promoting stem cell niche formation andhelping them to position in a right domain. But there is another there are some repressionsof this process. And those repressor need to be repressed to ensure that a proper stemcells are maintained and proper stem cell niche are positioned. And one of them arebelongs to another class of transcription factor which is called homeobox transcriptionfactor. And this you can see here that if you lookthe mutant of SERRATE gene, SERRATE is a nuclear zinc finger protein. In this mutant what youobserve that that the proper embryogenesis is totally defective, and there is no properorganization of the cells, there is no proper organization of them apical meristems. Butwhat happens that if you combine this mutant, serrate mutant with some of the homeobox geneslike PHABULOSA and PHABULOTA , these are the homolog genes. If you make double mutant ourtriple mutant, what happens that this defect of serrate mutants is complemented. What doesit mean? It means that somehow they are genetically interacting and they are basically suppressingthe phenotype of serrate mutant. How this happens? So, if you look the expressionpattern of this box homeobox gene PHABULOSA, so you can clearly see this is very symmetricalasymmetrically expressed or localized. You can see it is only present in the apical domain,it is not present in the basal domain or root apical meristem domain. This is important.So, this gene has to be repressed in the root apical meristem for proper meristem function.But what happens in the serrate mutant? In serrate mutants this asymmetric expressionpattern or transcript localization is lost for the PHABULOSA genes. Now, you can seethe PHABULOSA is expressed everywhere in the embryo and that is disturbing the patternthat is not allowing a proper root apical meristem to take place. But when you combinethis when you knock out this transcription factor in serrate mutant background, earlythere might be not a much complementation, but later stages this basically complimentthe phenotype.As you can see through the marker, so this is a SCARECROW marker SCARECROW just now youhave seen that it express in the QC as well as the endodermis initials. This is WOX5,we will more talk in detail in the in the next slides. But here just to know mark herethat this key WOX5 is another homeobox protein, but which expressed in a very specificallyin the QC cells. But what happens in the serrate mutant, both the markers lost their expressionpattern, which means that there is no proper QC identity, there is no proper stem cellniche organization and positioning. But when you combine, when you make a doublemutant, triple mutants, what you can see that they reappear their expression domain reappearin a right domain which means that now if you mutate PHABULOSA, PHABULOTA , there basicallythis mutant the serrate mutant or the triple mutant, they can reorganize their stem cellniche root stem cells niche and that is why they can rescue the phenotype.See if you look this model, this PHABULOSA protein which is very which is highly expressedin the apical domain, and it is kept repressed in the basal domain. At this state particularlyin root apical meristem, this needs to be totally kept repressed. So, there are twokind of mechanism. So, if you want to maintain root apical meristem, then some activatorhas to be activated, at the same time the repressor has to be negatively regulated toensure proper development.Another important regulatory pathways which is regulating root apical meristem maintenanceis WOX-CLE-ACR4 module. So, we will go one by one. What are the interaction? If you recallour previous few classes where we have discussed in detail, what kind of approaches we aretaking to study. Now, here what you are going to learn that how those approaches has beenused and how the data has been interpreted to understand a particular developmental pathway.So, this WOX5; WOX5 is another homeobox domain containing transcription factor, but it hasmore positive role on the stem cell, root stem cell maintenance or QC identity unlikethe PHABULOSA and PHABULOTA . If you look the expression pattern, so this is WOX5 promoteractivity or RNA you can clearly see that it is very specifically expressed in the QC.And if you look this is wild type, so this is QC. Just below the QC you have columellastem cells and then columella, but if you look in the wox5 mutant background this isthe QC, but the cell just below the QC, it looks defective, it is not normal cells. Andif you check the markers, different markers of the QC, for example, QC 184, you can clearlysee that though QC identity is not completely lost here, there might be maybe some of themarkers are disappearing. For example, QC 184 is disappearing, but WOX5 marker is stillactive. So, there is a defect in the identity. But another important thing if you look herein the wild type thesecolumella stem cells identity is basically here lost and it hasbecome more kind of differentiated. So, this has accumulated the starch granules, whichtells that WOX5 is important for stem cell maintenance for columella stem cell maintenancein the root.Then there is another peptides, which is called CLE peptides CLE40 genes if you look thismutant. So, this is wild type in wild type, typically you have QC then you have one layerof D 1 layer which is your columella stem cells, and then next layer is your columellacell differentiated columella cells. But if you mutate CLE40 genes what you see that thereis more than one layer of stem cells columella stem cells which means that in cle40 mutantsthere is a gain of stem cell activity. So, there is more than the required stem cellsactivity. And but if you take this wild type and treatexogenously with CLE peptide, CLE peptide you can synthesize and then what happens thateven the one layer of initial cells or stem cells has disappeared in wild type. So, inwild type, there is a endogenous CLE40 signalling is active, now you are putting more CLE40protein from exogenously. So, it might be working in the dose dependent minor. And ifyou have more doses, even one layer of this activity is lost.But if you look here when the endogenous CLE40 pathway is lost because of the cle40 mutant,now you are supplementing CLE40 from outside it basically restores the phenotype to thewild type, here you can see only one layer of columella stem cells. And these are justthe marker expression pattern for WOX5 RNA and QC 184 RNA. So, in this way, you can saythat WOX5 is activator or positive regulator of stem cell activity, whereas CLE40 mightbe negatively regulating the activity but we will see how exactly this is happening.Then if you combine this mutant, if you look the wox5 mutants as you see earlier in wox5mutant, you do not have columella stem cells. But when you treat with CLE40 proteins eventhe QC got differentiated. So, apart from the columella loss of columella stem cells,even QC has lost has accumulated the grain, so there is a gain of differentiation.Then there was another CLAVATA like genes which is CLAVATA2. Maybe we will see in theshoot when we will discuss in the shoot the similar kind of mechanism functions in maintainingshoot apical meristem. So, if you look clavata2 mutant; CLAVATA2 mutants basically it doesnot have any phenotype it looks very similar to the wild type Columbia.Then if you go again and look another mutants or if you see the genetic interaction withother genes, then what you can see there is another CLAVATA which is called CLAVATA1.In clavata1 mutant, this shows a phenotype which is very similar to loss of CLAVATA40.And another gene which is called ACR4, which is basically ARABIDOPSISCRINKLY4. So, thisalso phenocopy clavata40 mutant phenotype. Which means that and another important thingif you treat these mutants with the CLAVATA basically in clavata1 the the phenotype islost in fact it is more differentiation has started.But if you look in acr4 mutant, even after CLAVATA40 treatment, there is two layer ofcolumella stem cells. This suggests that they are working through the same pathway and CLAVATA40is upstream of ACR4. So, it is basically CLAVATA40 is working through the ACR4. So, if you lookhere this is CLE40 and then downstream is ACR4. So, if you do not have ACR4, even ifyou provide CLAVATA40, the phenotype cannot be rescued. So, ACR4 is required for CLAVATA40to work here. Then if you look again these are the doublemutant phenotype. So, this is cle40 alone there is two layers. When you combined clavata1and acr4, there is two layer. clavata1 and cle40 - two layers; clavata4 and cle40 - twolayers. So, all are having similar phenotype which suggest that all of these might be workingthrough the same pathway same genetic pathway.And then if you see the expression pattern of them, you can find this way. So, this greenis ACR4 expression pattern, and red is CLAVATA1 expression pattern, and this is the CLE40expression pattern domain. And another important thing what has been seen that this ACR4 isvery specifically localized to the plasmodesmata. Plasmodesmata in some of the future classwe will see that plasmodesmata is a channel through which lot of cell to cell communicationtakes place during the plant growth and development.So, here if we combine all these information together, so what you can suggest here isthat CLAVATA40 which is present here. This CLAVATA40 is actually the biochemical natureof CLAVATA40 is known, this is a kind of signalling peptide which works as a ligand and then theseare the receptors. So, this CLAVATA40 is received by ACR4 andCLAVATA1. And then this signalling is very important to restrict the domain of WOX5 toQC, so QC should not expand. And this is very important to ensure that a position of QChas to be fixed at a particular place. And once you fix the position of QC, eventuallyyou are going to regulate or positioning of entire stem cell niche.So, if you look here carefully, so there are multiple pathways PLETHORA mediated pathwaySHORTROOT SCARECROW mediated pathways, and then WOX5 ACR4 CLE40 mediated pathway, allof them working together to ensure proper root apical meristem maintenance and the function.Apart from these regulators, plant hormones which are very very important in growth anddevelopment, almost they regulate every aspects of growth and development auxins, cytokinins,Gibberellic acid all these hormones are very very important. So, here if we look we maynot go in a very very detail, because this is a very complex process of hormonal signalling,but something to say that auxin, brassionosteroid all these hormones are also playing very veryimportant role either directly or through the same genetic regulatory pathway whichjust now we have discussed to ensure the proper root apical meristem maintenance.For example, if you look here this is SCARECROW. SCARECROW is negatively regulated by or repressedby the cytokinin this is response regulators which works in the cytokinin responseprocesses.And then this is auxin; auxin is activating some of the auxin response factors. And theseauxin response factors are regulating the activity of WOX5. Auxin is also regulatingthe activity of PLETHORA’S. So, basically if you look in this picture,you can see that there is a cross talk between auxin and cytokinin together which ensuresa proper root apical meristem and proper meristematic activity and a critical balance between celldivision and cell differentiation during growth and development. And then this is a very complexpicture, we will not go in the detail.