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Welcome to Plant Developmental Biology.So, in last class we started studying Root Developments.So, we will continue that.So, this slide you would have already seen in previous lectures.So, this is just to remind that there are three major steps which takes place duringthe process of root development; the root apical meristem, tissue patterning and rootbranching which is basically lateral root development.So, in last class what we discussed more towards how root apical meristem is maintained.So, this is very important to produce or to generate a large number of stem cells.So, this is just this picture you have you would have already seen in the last class,but this is just to recap you before going further transition.So, it is very important to have a group of cells which should always divide and theygive rise to the cells which is required for the process of differentiation.And the first and very important thing what happens for the maintaining apical root meristemis positioning stem cell niche.So, this is the region which is stem cell niche which means that the cells which arepresent in this domain, they are stem cells, they have stem cell property, they can divideand they can provide cells for later stage of differentiation and their position is relativewith the QC.So, the cells which are in direct contact with the QC they remains as a stem cells,but next step what happens is the differentiation.So, what happens that these initial cells they depending on their position, they taketheir daughter cells after the cell division take different identity.So, once this cells divide what happens let us assume if this is the cell if this cellsdivide here.So, there are two daughter cells the daughter cells which is towards the QC it remains asa stems cells or as initial, but the daughter cell which is away it enters in the processof differentiation.And just now I said that the fate of this cells the differentiation program in thiscells will depends on the relative position of the cells.So, for example, if you look here.So, there are few things which I just want to remind this is the initial which is a calledCEI which is initial for cortex and endodermis.So, you can see here both the layers cortex and endodermis they are originated from thesingle cells; similarly this initial is for epidermis and lateral root cap how this happenswe will see later these initials are for columella cells they give raise to columella and theseare the vascular cells initials they give raise the vascular tissues.And once this process of differentiation starts one thing is very important, if you take thisseed if you take any plant and germinate it at very early stage, as you can see this isat the embryogenic state the pattern is defined, but at early stage may be the rate of celldivision is slightly more relative rate of cell division is more than the cell differentiation,but at certain time point there has to be a balance between rate of cell division andrate of cell differentiation.This is extremely important to ensure that a proper amount of meristematic cells aremaintained at the same time sufficient amount of cells required for the differentiationshould be provided.One and very important step of differentiation is asymmetric cell division.You can say this is the first step in many of the differentiation program in the lifecycle of plant development, if you recall your embryogenesis.So, even the first cell division of zygote is asymmetric in nature.Asymmetric means producing two daughter cells which are not exactly similar.So, if you look here again this is the same picture, but what you can see.So, we can see that these are the initials which is for cortex and endodermis and so,cortex and endodermis if you look the first step of differentiation.So, after the cell division so the cell division will generate two cells the cells which isnow in contact with QC will remain as CEI, but this cells will undergo the process ofdifferentiation and what happens once the differentiation process start its undergoanother round of cell division, this is the cell division and now this cell division isdifferent in nature.So, there is a reorientation of the plane of cell division.So, this cell division if it is periclinal this is called anticlinal and this cell divisionessentially generates two layers from here onwards.One layer which is going to give endodermis as you can see here and the outer layer whichis going to make the cortex.Similarly if you look the initial this initial is epidermis and lateral root cap initials,this initials again undergo first round of cell division after cell division the innercells the basic and inner cells which are again in contact or which is a part of thestem cell niche will remain as a stem cells.But other inner layers this start another cell division and give raise to epidermiswhereas, outer cells it will undergo the process of a specific cell differentiation programto give lateral root cap this is important.Third initials if you look these are the columella cell columella initial cells this columellainitial cells again undergo the process of cell division and they remain the stem cellsproperty in the cells which are just below the QC, but the cells which are distal fromthe QC they undergo the process of columella specific differentiation program.This is what happens in the vascular initials; vascular initials based on their positionyou can say they can be pericycyle initials, they can be protoxylem initials, metaxyleminitials and procambium initials or phloem initials.So, in all this case what you see that the process of differentiation start at a firstasymmetric cell division itself, then it undergo lot of programming lot of a cell divisionand cell differentiation programming together and lot of genes which are getting regulatedor which are getting activated they are regulating these process.So, what we will do we will take few of the example and we will try to discuss how thishappens.So, if you take the differentiation of cortex and endodermis and what happens that, justnow we said that cortex endodermis initial cells it undergo the process of asymmetriccell division, but what are the regulators?So, if you recall your previous class you remember that these two proteins are veryimportant SHORTROOT protein and SCARECROW protein, they help in regulating not onlystem cell maintenance, but they also help in giving a specific identity to endodermis.And what happens that these SCARECROW protein it has been found to physically interact withRETINOBLASTOMA like protein and what and this if you look SCARECROW protein is also interactingwith SHORTROOT and interacting with RBR, but their interaction is totally different andthese three important residues ACA it is important for interaction of SHORTROOT protein withRBR if you mutate this residues at the place of this residues.So, this is actually mutant if you mutate it what will happen that, the SCR will loseinteraction with RBR, but interaction with SHORTROOT protein is not disrupted.And how these regulates we are going to look here.So, this is a schematic diagram which we have already discussed.So, if you look this picture what happens that here you have SCARECROW promoter drivingSCARECROW:YFP fusion protein in scarecrow-4 mutant background.So, this is a close to basically wild type you have scarecrow mutant, but this mutantphenotype is getting complemented by YFP fused SCARECROW protein, but if you mutate the interactionbetween SCR and RBR what happens that you can start looking some phenotype?And one phenotype you can see here is that, there is a extra layer formation.So, normally in wild type from CEI only two layers are formed and in this two layers theouter layers always remains as a cortex and inner layer differentiate into endodermis,but here if you look in this disrupted interaction mutants what is happening?This is more clear on the upper side if you look there is.So, this is wild type you have only one cortex or equivalent wild type and you have onlyone endodermis, but when you mutate these residue when you disrupt this interactionbetween SCR and RBR you can see that between cortex and endodermis there is extra layer.So, this tells that this interaction between SCR and RBR is very important in regulatingcell division pattern.Another important thing what you will look here that this is one very interesting genewhich is called CYCLIN D6 gene and if you look its promoter fusion construct this genevery specifically expressed in the daughter cells of CEI.So, when CEI undergo the first round of cell division you can see this genes are expressedthis CYCLIN D gene is expresses, but immediately after that it its expression is totally disappearing.So, which means that there is a possibility that the SCR RBR or this genetic regulatorsare activating expression of this which you will look later on in detail, but this mightbe interesting.Because when you disrupt this interaction between SCR and RBR what you see that, thereis a extra layer and more important that expression of CYCLIN D6 expands.So, it expands in both the layers.So, this tells that this interaction is not only important in maintaining the number ofcell layers, but it is also important in restricting or in regulating the expression domain ofCYCLIN D. Another important thing if you look here.So, this is plant treated with auxin or you can increase the auxin level when you increasethe auxin level, auxin is hormone plant hormone I have already told in previous class thatauxin is very very important it regulates lot of process.And here also particularly in roots auxin has a very very important role to play andwhat happens, if you induce this plants with auxin you can see there are extra layers andthese extra layers have an induced expression of CYCLIN D.But important thing here is that in this extra layer out of these extra layers your endodermisis only the inner most layer as you can see from the localization pattern of SHORTROOTprotein, which is also always in the endodermis.Another important thing here if you look.So, this is SHORTROOT protein complemented with SHORTROOT GR fusion.So, if you recall one of our class we have discussed that glucocorticoid receptor fusionto a transcription factor basically make it inducible in a way that when you treat withdexamethasone then and only then this protein will enter in the nucleus and do its function.But if you do not induce then the protein is there, but protein cannot go inside thenucleus and since it is a transcription factor and if we it does not enter in the nucleusit will not perform the function.So, what happens here?So, if you look here.So, in shortroot mutant since this mutant we are not treating with dexamethasone thatis why you do not see the complementation.So, this is a kind of typical shortroot protein mutant and you do not see induction of expressionof CYCLIN D gene.But when you treat with dexamethasone; dexamethasone is basically allowing SHORTROOT protein toenter inside the cell and when SHORTROOT protein is entering inside the cell you can see CYCLIND expression starts which means that SHORTROOT protein is activating expression of CYCLIND. And when you treat dexamethasone plus auxin, then you can see that it is mimicking thisphenotype where you have extra layers and extra layers are basically increasing havingmore or expanded expression of CYCLIN D. So, this all together suggest that SHORTROOTSCARECROW is important and it is working to activate auxin dependent activation of CYCLIND proteins and SCR’s interaction with RBR is also all these regulative networks is veryvery important in regulating.the asymmetric cell division of CEI and eventually a layer of endodermis and cortex formation.So, if I summarize whatever we have discussed here.So, there are two things to remember one is that this SHORTROOT protein which basicallyin some of the future class you will know how exactly it moves, but SHORTROOT proteinit move from one cell to another cell and this movement is essentially generate a kindof gradients.So, you can see the cells because you know SHORTROOT proteins are produced in the vasculartissue, the cells which is very close to vascular tissue it is having more SHORTROOT and thensimilarly the auxin signaling path way.So, what happens in the auxin signaling path way?Auxin is synthesized in usually highly dividing cells or in the young tissues and then thereis a well established system that the auxin can be transported in a very specific manner.So, there is a process called polar auxin transport.So, auxin can be synthesized somewhere, but it can be transported to different placesand this transportation basically ensures auxin concentration and what happens thatthis basically generates a kind of gradient in the auxin concentration, it generates maximumamount of auxin in some tissues minimum amount of auxin in another tissue and auxin workslike a morphogen.So, if you have high amount of auxin it can induce a very specific genetic program atlow amount of auxin, it can perform a different function.So, the combination of all this kind of things if you look here.So, what happens in the position A?So, this is your position A is basically CEI in CEI we have high amount of auxin and thenwhat happens that SHORTROOT and SCARECROW activates CYLCLIN D in auxin dependent mannerand this process essentially induce asymmetric cell division and after asymmetric cell divisionif you look in the inner cells what happens the domain of SHORTROOT and SCARECROW is restrictedonly to the endodermis cells and how it is restricted is another different regulationwhich we are not going to talk here, but what happens that in inner cells since auxin levelis going down.So, you have SHORTROOT SCARECROW mediated cell fate determination of the endodermis.But overall what happens the combination of auxin SHORTROOT SCARECROW RBR basically ensuresthe first or the asymmetric cell division in CEI and then eventual process of layerdifferentiation.Similarly, similar kind of cell division is also very important in differentiation ofepidermis and lateral root cap.So, if you look this is basically epidermis and lateral root cap what happens that theinitials first undergo the process of pericycle cell division and then it undergo the processof anticlinal cell division and this essentially results in formation of epidermis and lateralroot cap.So, the origin of both the layers are from the same initial and there are two transcriptionfactor both are NAC domain containing transcription factor one is called FEZ another is calledbasically SMB which is SOMBRERO and what happens that in mutant background what you can seehere the frequency of periclinal cell divisions significantly decrease.So, this is the wild type level this is fez mutant and this is smb mutant.So, which means that both are very important for making periclinal cell division and ifyou look in the mature root what happens that in wild type and this has the eventual effectlyon the lateral root cap development as you can see here that in wild type there are fourlayers of the lateral root cap, but in when you have fez mutant you have only three whereas,in smb mutants the number of cap layers are increase.So, basically they are regulating lateral root cap development in a different manner.If you look here how columella initials undergo the process of differentiation the same regulatorshas been shown to play very very important role.So, if you look just the expression pattern of FEZ and SMB protein what happens in theduring embryogenesis the FEZ start expressing here at that stage SMB was not activated,but slightly later stage SMB is getting activated and when you overexpress basically this FEZprotein what happens in endodermis.So, you are using SCARECROW protein.So, you are over expressing FEZ protein and what you can see there is additional roundof periclinal cell division.So, this basically supports the previous observation that FEZ is important for periclinal celldivision in mutant that cell division is decreased but when you overexpress there is additionalcell division takes place.But if you look the expression pattern and their regulatory network it is very important.So, if you look this FEZ; FEZ is not expressed in the QC, but it express in the initialswhich is columella initials and then this expression when this columella initials undergothe process of cell division what happens?After this cell division the expression of FEZ remains in the lower cells, but it totallyand immediately very quickly disappears from the initials which are close to the QC.And SMB induction starts only in the lower layer which is your initial daughter cellsof the initials.So, if you look all these expression pattern and mutant phenotype if you combine thereis a regulatory mechanism what happens that at the very early stage FEZ is getting activatedFEZ is getting activated in these cells then this initial cells undergo the process ofcell division.When cell division occurs in the lower cells FEZ basically activates the expression ofSMB, but this SMB once SMB is activated it immediately repress the FEZ expression andthis how there is differential expression pattern and this basically regulatory networkensures a proper stem cell identity for the columella stem cells and the distal cellsto enter in the process of differentiation proper differentiation program.If you look more detail how auxin regulates root cap formation there are other evidence.So, basically these are AUXIN RESPONSE FACTORS.So, AUXIN RESPONSE FACTOR are class of transcription factor which basically regulates the processdownstream of auxin signaling.And what happens that if you mutate AUXIN RESPONSE FACTOR in single mutant you do notsee much phenotype, but when you make a double mutant of AUXIN RESPONSE FACTOR 10 and 16what you see that there is a defect in the root growth and if you look there basicallycolumella what happens in wild type you have maturate, or differentiated columella butin this mutant background most of the columella cells are not differentiated.So, there is a problem in the differentiation they are more kind of dividing and layersnumber of layers are also increased.Another important thing is that this AUXIN RESPONSE FACTOR 10 and 16 they are regulatedby micro RNA 160.So, micro RNA you know that they are small RNA and micro RNA what happens that if theytarget a gene they silence its expression.So, what happens this you can see if you overexpress micro RNA basically this gives a phenotypevery similar to the loss of function of the AUXIN RESPONSE FACTORS.So, when you overexpress MIRNA there is more micro RNA more micro RNA more down regulationof AUXIN RESPONSE FACTOR which means that it will have a similar phenotype as the auxinresponse factor mutant.But this is interesting if you.So, basically micro RNA what happens?These are just 21 to 24 nucleotide molecules and they have some complementarity sequencewith the target and if you mutate this target binding sequences this overexpression of microRNA and then you complement with the mutant version of auxin response factor you do notsee the phenotype which suggest that the micro RNA is regulating AUXIN RESPONSE FACTOR 10and 16 through the binding and this regulation is basically important in regulating columellacell differentiation or root cap formation.Similar kind of phenotype you can see here.So, if you have wild type this is the differentiation pattern if you treat with auxin.So, here there was loss of auxin now we are having gain of auxin, when you have more auxinyou can see the differentiation even stem cell properties are lost.you have this YUC, yuc mutant is basically defect in auxin biosynthesis internal biosynthesis of auxin is reduced.So, basically this is low amount of auxin you can see two layer of stem cells.Similarly, if you look this mutant background and if you look the complementation or ifyou make a double mutant triple mutant auxin response factor 10, 16 if you combine withthe wox5 all this kind of things you can see here that these AUXIN RESPONSE FACTOR, microRNA, auxin they all are working through the WOX5 path way which means that the combinationis not basically helping in detail.So, another there are some more important mutants which are playing may be or the geneswhich are regulating later stage of root cap formation.The final stage of root cap formation basically is programmed cell death because the at bythe process of program cell death these caps are basically removed at a certain frequency,but if you look this mutants background what happens that this lateral root caps are notgetting removed.So, these are basically process of root cap maturation this is also important and thereare some another transcription factor has been identified which regulate this process.So, if I combine everything together and if we look in a overall regulation of the transitionbetween cell division and cell differentiation.So, what happens that auxin is regulating ARF 10, 16 and these are working through theWOX5 path way to restrict or to regulate the stem cell niche and allow a proper differentiationprogram.Here if you look layer wise how regulation is working.So, this is your QC this is c 1 c 2 c 3 c 4 c 5 and this is here.So, what happen in the QC?In QC basically there is a less division less differentiation or you can say QC is no divisionno differentiation.So, but WOX 5 is present, but if you come one layer down this layer is basically stemcells; stem cell has to be divide.So, here division is dominating over the differentiation and what happens WOX 5 is very active RBRis active and FEZ is active.But in other layers c 2 to c 5 layer what happens ARF 10 ARF 16 SMB FEZ and RBR theybasically interact and they basically shift the transition from cell division mode tocell differentiation mode and if you come in the last layer which is called terminaldifferentiation, the final layer differentiation there is another gene which is called BRN1and BRN2 and SMB they basically initiate or activate the process of terminal differentiation.And that is why if you do not have BRN1 and BRN2 that if you remember this is the terminaldifferentiation is defective and that is why you can see multiple layer of lateral rootcap formation.So, we will stop here and in next class we will discuss vascular tissue development.Thank you very much.