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Welcome to Plant Developmental Biology course.In today’s class we are going to discuss Root Branching particularly Lateral Root Developmentin plants.So, branching is very important or very critical events in the process of plant growth anddevelopment, because this helps in establishing a proper architecture in the plant body.If you look a typical model plant here, you can see that the root system has some branchingwhich is we call basically root branching and then shoot system they also undergo theprocess of branching.In today’s lecture we will focus on root branching.And this is very important root branching basically increase the surface area of entireroot system, it establishes a proper root architecture and which is very important forplant to be efficient in absorption and anchorage function of a plant.So, if you recall previous lectures.So, what we have discussed?We have discussed the meristem maintenance which happens at the tip of the meristem,then if you come slightly higher side during the stage of cell elongation and maturation,these are the stages or these are the places where tissue patterning occurs where differenttissues, different cell types, different identities are taken place, but if you come in the maturationzone or in the differentiation zone the branching basically visible branching start.So, we will cover this root branching in this lecture.So, root system architecture as I said is very very important in the plant species andthere is a huge variation in the root system architecture in different plants.But if you divide the total kind of root types based on the architecture, there are two majorroot types one is the tap root system another is fibrous root system.In tap root system you have one dominantly primary roots which you can see here thisis typical Arabidopsis root system this is carrot, but fibrous root system is it is presentin the monocots whereas, tap root systems are present in dicot plants.In fibrous root system there are primary roots, but later on there are some additional rootswhich starts coming in these systems which are normally called adventitious root or crownroots depending on the species.So, this is a typical example of maize and rice; in rice you can see here these are thecrown roots, this is the primary roots and then lot of lateral roots which are comingfrom both primary roots as well as the adventitious root this is the case of maize.So, the important thing here is that how the development or pattern or how this root systemarchitecture is established in plant is a very critical, very important.So, we will take here example of root lateral root development and mostly in model dicotplant Arabidopsis thaliana.So, if you look this is Arabidopsis thaliana seedling, you can see here this is the primaryroot.Primary root is growing and then if you look in the base in the lower zones you do notsee any visible roots because these are the regions where your meristems.Meristem is somewhere here there you have elongation zone and, but here if you comehigher side, you can start seeing that lot of emerged lateral roots has come.So, these are the lateral roots which is coming in the maturation zone, but as I said theprocess or the program; the developmental program which is required for formation ofthis lateral root has started somewhere here.So, this is very important for us to understand.And if you look in Arabidopsis the process of lateral root development is well establishedand it is known that the cell so, basically what is happening here if you look this allbranching this happens post embryonically.So, lateral roots are coming from the root.So, they are root borne root which means that you have already a differentiated root system,then suddenly a new branches or new roots are originated from that root.And this is only possible if there is new meristems which has been generated somewherehere in the root which is kind of lateral meristem.And which means that there is a process.So, you have a differentiated cell then the differentiated cell has to undergo the processof cell division and then it will generate a kind of meristem, and then that meristemwill start the process of development.In Arabidopsis it is known that the cell which is next to the xylem the pericycle cell.So, this is basically longitudinal view.So, you can look here this is epidermis, then below epidermis you have cortex then belowcortex you have endodermis, and then below endodermis inside endodermis this layer iscalled pericycle and then you have the vascular tissue.And it is known that since it if you look a cross section I do not have any picturehere, but if you make a cross section what you will find that this pericycle layers arelike this.But, the xylem makes a kind of axis here.So, there are pericycle cell which is close to the xylem is called xylem pole pericyclecells.And it has been seen that in case of Arabidopsis thaliana these pericycle cells basically havecompetency and potential to initiate lateral root developmental specific program.And when this happens; what happens that, this mature or differentiated pericycle cellsthey undergo the process of cell division.So, there is; so, differentiated cell undergo the process of dedifferentiation then startdividing and then start redifferentiation of the lateral root.So, this entire process of lateral root development can be divided into different stages.So, at very early stage what is happening there is one pericycle cells, when it startscell division then more cell division and there are different if you look the orientationof cell division some are periclinal and some are anticlinal, and these results in growthof the primordia this is called lateral root primordia.And then at later stage of the lateral root development different tissues start takingtheir identity like a typical root and here again if you recall; if you remember the QCand root meristem organization.So, here is new root meristem which is established here which has QC then you have epidermis,you have cortex this all the same layer of the root system.And once this primordia start growing, then it will rupture and it will start coming itwill come out.So, this is called root emergence.So, the entire process of lateral root development can be divided into different stages; thefirst stage is the root meristem lateral root specification primordia specification, thenthe primordia differentiation where all this tissue patterning is happening and then finally,lateral root primordia emergence.And one important thing which has been found that this process of lateral root initiationstarting from the initiation, starting from the specification till the emergence planthormone auxin has been found to play a very very important role in this entire process.And if you recall some of the previous class we have seen that basically auxin functionsas a morphogen it functions in the gradient dependent manner or the concentration dependentmanner.So, if you have high amount of auxin and there is a full mechanism in plants which basicallytransport auxin.So, you can relocate auxin from one cell to another cell and this kind of mechanism iscalled polar auxin transport and this polar auxin transport basically helps in generatingauxin maxima in some of the cells and auxin minima in another cells.So, this is what happens.So, if there was a correlation if you look the auxin activity you can see that, thesecells they start auxin maxima.So, these all cells if you look even in these cells, the cells is this cell is undergoingthe process of asymmetric cell division and the cell the smaller cell which is towardsthe centre they are having more auxin than the cells which are outer side.And then eventually in the development, the auxin maxima started getting accumulated onlyin this region very tip of the were the developing rootsSo, we will see this mechanism in details.So, this is just to show that lateral root primordia establishment initiate from xylempole pericycle cell.So, this is J0121 is a marker which very specifically express in mature or differentiated pericyclecells xylem pole pericycle cells.So, if you look here this is driving GFP.So, if you look very carefully this is the xylem cells, you can identify xylem cellsbased on its structure because xylem has lot of modifications, lot of lignin and secondarycell wall modification.So, based on that you can clearly identify this is a basically xylem and the cell whichis just next to the xylem is pericycle cell; xylem pole pericycle cell.And these xylem pole pericycle cells is very specifically expressing this gene, and whathappens once these xylem pole pericycles they start the process of lateral root initiation.So, if you look here in this xylem pole pericycle cell has started dividing at a particulartime point and once it has started dividing, it is basically losing this identity of pericyclecells and it has acquired stem cell identity.And that stem cells eventually going to give rise to the primordia at early stage and thisis emerging primordia; emerging lateral root primordiaThis is cross section.So, basically this is driving GUS expression under J0121 promoter all of these and if youlook here if you make a cross section you can very clearly see this is the xylem axis,this is phloem pole and these two cells which are next to the xylem axis or which are incontact with the xylem axis, they are xylem pole pericycle cells they are very specificallyexpressing these markers.Another way to test whether that xylem pole pericycle cells are directly involved in thelateral root development this experiment was performed.This DTA is basically it produce a kind of toxin.So, if you produce this toxins in a cell, the cell will be killed and then you see whatis the effect.So, here what is happening that these toxins are being produced very specifically in thesecells which is xylem pole pericycle cells.And if you do that what you can see that in these plants when you kill the xylem polepericycle cells, you do not see lateral root development here.So, this also suggest that flow; xylem pole pericycle cell is the cell which gives riseto the lateral root developmental program ok.So, as I said that auxin is very very important hormone plant hormone which undergo the processof; which regulates almost every aspects of plant development.So, maybe here we will just have a quick look of auxin signalling how it happens.So, auxin; as I said auxin is a very important plant phytohormones which is being synthesizedmostly in the young tissues.And when this auxin is synthesized, there are lot of auxin bio synthesis pathway 3-4;4 auxin biosynthesis pathway, then you have lot of enzymes which are helping in auxinbiosynthesis and once auxin is biosynthesized there is a mechanism to basically maintainits homeostasis.So, auxin; high amount of auxin is not good for some of the development.So, what happens that if you have high amount of auxin, then auxin can be stored in formof inactive form.So, there is a mechanism that you can inactivate auxin that helps in maintaining auxin homeostasisin a particular cell.And then the active pool of auxin which is basically able to initiate auxin signallingpathway can be distributed very properly through a process of polar auxin transport.So, through this process you can basically distribute this auxin to a cell where auxinsignalling has to be initiated.And what happens, and if you look the signalling.So, what happens?Auxin, Let us assume that a cell has auxin.So, auxin is basically received by receptor; auxin receptor which is TIR1 here, but thereis a mechanism let us assume in a cell.If there is no auxin what happens?There is a class of transcription factor which is called auxin response factor.Auxin response factor is a class of transcription factor which regulates set of genes whichare working downstream of auxin signalling pathway or you can say that auxin signallingpathway is regulating expression of many genes.And what happens if there is no auxin there is a very high amount of negative inhibitorof auxin response factor which is Aux/IAA.So, essentially Aux/IAA it can; so, basically it can pair with auxin response factor andit inactivate auxin response factor, it does not allow auxin response factor to go andactivates the genes which is working downstream of auxin signalling pathway.But if you have auxin; if you have high amount of auxin, the auxin is basically receivedby the receptor and this receptor basically the complex of receptor and auxins targetthis Aux/IAA protein for degradation.So, when there is auxin this protein will be degraded through a mechanism and then oncethis protein will be degraded the auxin response factor will be freely available.Now, this auxin response factor as I said it is a transcription factor it goes and itbinds to auxin response element and then activate the gene or repress the gene depending onwhat is the nature of this auxin response factor and result is auxin responses.So, there is these two construct which are very extensively used in plant developmentalbiology.So, what has been done?So, basically a synthetic promoter has been created where the auxin response element thisis the element or DNA elements where auxin response factor can come and bind.So, this has been cloned along with a basal minimum promoter and then this construct DR5or DR5rev this has been generated.So, essentially a synthetic promoter is generated where we have put a basal expression elementpromoter, basal promoter along with auxin response elements.So, which means that this protein will have a very low or very basal level expressionunder normal condition, but if there is auxin response factor if it comes and binds to theauxin response element, it can enhance the expression.So, this basically system this is called DR5 based reporter system it basically reportsauxin responses.So, if you see high amount of signal or if you see high amount of reporter activity;DR5 reporter activity which means that, that is the cell where there is a auxin signallinggoing on.This is a typical example if you take this construct and check in the root.So, this is the cap root cap so, you can see very high amount of signal root cap.And then reporter gene you can choose as per your choice, here the reporter gene is GFP,here is the nuclear localized YFP or VENUS, here you have endoplasmic reticulum localizedRFP.So, based on that you tell exactly where is the auxin responses.So, as I said that auxin signalling is regulating lateral root development.So, what is the mechanism, how it regulates and what is the proof that auxin signallingis regulating lateral root development?You can look here so, if you recall previous slide I said this IAA it is a kind of oneAux IAA; Aux IAA basically is a negative inhibitor of auxin response factor.So, if you have high amount of Aux IAA it means that no auxin signalling will be activated,and here this is the mutant.In this mutant what happens that the level of this protein is stabilized; so, you havehigh amount of Aux IAA.High amount of Aux IAA means in this mutant there is no auxin signalling activated; noauxin signalling activated results in no lateral root formation.So, this suggests that activated auxin response signalling is important for lateral root formation.Now these are two auxin response factor, auxin response factor 7 and auxin response factor19, if you look double mutant here you can also see that there is no lateral root formation.So, even auxin is there, auxin signalling is started so, again I will come here so,auxin is there this protein is degraded, but this auxin response factor is mutated.So, if you do not have auxin response factor, you cannot generate auxins response, and whenthere is no auxin response you cannot initiate lateral root developmental program.This you can see here as well and then it was also identified that this auxin responsefactors are regulating some other class of transcription factor, these are LOB domainor LBD domain containing transcription factor.As you can see here that if you take this auxin response factor 7 and 19 mutant andput LBD 16 you can already rescue the phenotype.So, there is lateral root formation.So, which suggest that auxin is basically negatively regulating a negative regulatorof auxin response factor.So, Aux IAA is basically negative regulator of auxin response factor.So, when there is auxin signalling auxin signalling is degrading this factor which means thatit is activating expression of auxin response factor 7 and 19 and then 7 and 19 is goingand activating LBD 16 18 29 and 16.And this signalling pathway once these genes are activated you can start lateral root initiationprogram.In this mutant background or in this experiment you can clearly see that though these genesare not here they are basically mutant, but you can you are providing downstream gene.So, you are putting LBD 16.So, if you activate this signalling from here onwards, you can already see the lateral rootspecific developmental program, which means that they are working in the same pathwayand they are regulating the process of lateral root initiation.Another important role what auxin plays here, it regulates the positioning of lateral root.So, it is not only the lateral root development, but where in the primary roots lateral rootshas to be that is also regulated by auxin signalling.So, if you look wild type growing Arabidopsis seedling here, you can see that the mode oflateral roots are alternates.So, one right left, right left, right left and in normal wild type this distributionis quite equal.So, you can see that around 50 percent lateral roots are coming towards left side, 50 percentsare coming towards right side.But if you have auxin mutant, auxin signalling mutant or auxin response mutant you can seethat this patterning is changed.So, now, you have more towards left less towards right.So, basically the balance is basically disturbed when you have the mutant background.And here this is again proofing same thing that auxin is important, this is a dominantnegative mutant which basically suppress auxin signalling pathway.So, if you over express this in xylem pole pericycle cells what happens that, auxin signallingis suppressed and you do not have lateral root development.So, suppression of auxin signalling very specifically in the xylem pole pericycles almost stop lateraldevelopmental program.So, this suggest that, the high amount of or activation of auxin signalling in xylempole pericycles cell is important for lateral root development.. This you can see here quite visibly so, thiscorrelate so, this is the DR5 construct just two slides before I discussed which is auxinresponsive construct, and if you take luciferase assay and if you look here there is a clearcorrelation.So, wherever you have high amount of signals it means that there is high amount of auxinsignalling there, you have lateral root primorida or lateral root development.And if you look the very early stage of lateral root development you can see here.So, this is DR5 GUS expression.So, basically this auxin or auxin signalling is getting activated very specifically inthese cells once auxin signalling is activated this cells start undergoing the process ofcell division there is cell division here, and then the process of primordia initiationsbegins.And then the process of initiation begins then auxin signalling is getting restrictedat the tips here and this process is normal pattern.And you can see here this is very interesting experiment which also proves that it is veryimportant to activate auxin signalling very specifically in xylem pole pericycle cells.So, basically here what you do, you use Cre Lox based random integration of a proteinor a gene which is responsible for auxin biosynthesis.And then you look for a line where this has been integrated in the xylem pole pericycle,this you can do because it has been combined with GUS.So, if you look this plant.So, you can see that these cells which is showing basically GUS activity it is xylempole pericycle cell, because it is next to the xylem and it has this iaaM expressingiaaM.This enzyme is basically responsible for converting tryptophan into auxin.So, what happens if you do not provide tryptophan even though the enzyme is present, but auxinis not synthesized.Then you see that there is an enzyme expressing here, but you cannot initiate lateral rootspecific developmental program.But when you add tryptophan what is happening that, these cells where this enzyme was presentit has got the substrate now it has produced auxin.And once auxin is produced in these cells, you can see that cell division starts andnumber of even primordia per seedling is increased significantly in presence of tryptan as comparedto in absence of tryptophan.Another interesting and important thing this is some of this we have already discussedthat, actually auxin starts working at a very very first step of the lateral root differentiation.Even at the stage of lateral root founder cells specification and this happens throughthe process of asymmetric cell division and this is the LBD 16 same LBD 16.So, if you look the process and this is nuclear localized signal, you can clearly see thatthese are the cells which are having high amount of auxin DR5 is basically telling theauxin activity, and the same cells they are expressing LBD 16.So, there is overlap.So, wherever auxin signalling is highs LBD sixteenths are expressed here.And if you look in the arf 7 and 9 mutant background you see LBD expression is disappeared.So, all this experiment basically suggests that auxin is at very early stays in the firstcell auxin is repressing this pathway and specifically activating this which is basicallystarting the symmetry breaking.The symmetry breaking is very important for lateral root initiation.So, if you have this is the specification of founder cell.So, what happens this is your pericycle cells, then in pericycle cells once there is auxinmaxima; auxin maxima basically what happens, you will see in the later slide that oncethere is high amount of auxin the nucleus start migrating towards the common pole.So, there is a process of nuclear migration.Now, you can see that this nucleus are coming very close to the common cell wall.And when they reach to the common cell wall, these cells basically asymmetrically dividelike this and then you have two small cells in the centre and then two cells which areoutside.And if you look the auxin responses, auxin responses is very high here and it is slightlyreduced in the lower cells ok.This is the process of for nuclear migration which is very important or you can say oneof the first step which is required for lateral root initiation program.So, you can see here.So, this is transgenic plant where three reporter markers has been expressed in the same plant.This reporter marker is marking the plasma membrane so, you can see every cell linesplasma membrane.Then you have H2B which is histone 2 protein, histone 2 protein basically it has RFP signaland it will localize to the nucleus because histone protein is nuclear protein, and thenyou have DR5 GFP which has basically auxin response and if you look very carefully here.So, what is happening?These are the cells.So, at very early stage you can see here that you have only red signal means you have nucleus,but there is no DR5 activity yet there is no auxin response initiated that is why youhave red cells.But slightly later stage this is time-lapse video what happens?At slightly later stage the color of this red started turning to the pink which meansthat in the same cells where in the nucleus of the cells auxin responses are getting activated.When there is auxin responses then you will have GFP signal and when GFP and RFP willstart merging the color will start changing from red to the yellow side.And then you can see slightly more later stage this becomes more yellow and this is the sitewhen you can also see that nucleus has also started migrating.So, here nucleus was very far from the this cell wall, but once auxin has auxin responseshas started in these cells, nucleus has started coming very close to this common cell wall.And then once it is very close, you can see that there is a cell division here a nucleardivision and cell division, and result is that you can see four cell stage and commoncell wall and just after that there is a cytokinesis and every cell has their own nucleus.So, this is the cell division.So, if you look here this is this tells that, there are few things which is happening verycritically and this process is regulated by auxin.The first thing is happening that there is high amount of auxin, auxin amount is goingup.Once auxin amount is reaching to the auxin maxima nucleus start migrating, nucleus iscoming close to the cell wall or cell wall then the cells are dividing, this is the veryfirst step of lateral look primordia initiation.And this process is not happening when you have auxin mutant; auxin signalling mutant.So, if you look auxin arf 7 and 19 you can see that there is no clear nuclear migration,even if there is nuclear migration it is not getting cell division is not occurring.So, this also proves that auxin is important for it.And then another gene which is called GATA23.GATA23 is another class of transcription factor and it was observed that it is showing veryoverlapping expression pattern with the auxin.So, you can see before asymmetric cell division it is expressed in the in the xylem pole pericyclecells, then during stage 1 stage 2 stage 3, but at very later stage the expression levelis going down cross section also proves the same thing.And if you look GATA23 promoter driving GATA protein fused with GFP you can see the geneis or the GATA23 is expressed even before nuclear migration and it continues duringthe nuclear migration.So, this could be one of the early regulator of nuclear migration or the process of LRfounder cell specification.This is also clearly visible here before asymmetric cell division you can see here the signalthen there is a nuclear migration and then at later stage there is cell division andall the cells are making the primordia.Another thing here if you look this GATA23 expression is induced by auxin.So, if you treat with auxin you see very high amount of GATA23 expression, but this inductionis not happening when you have auxin response factor mutant.So, this also tells that GATA is working downstream of auxin signalling pathway.So if you have auxin signalling pathway, auxin signalling pathway is activating GATA andGATA might be regulating something and this is clear here.So, if you have gata mutant, this is gata loss of function and this when you over expressingGATA23 in xylem pole pericycle.So, if you see different stage of primordia what happens?If see early stage of primordia when you do not have GATA23 you can see that number oflateral root primordia is decreased, but when you increase or overexpress it is increased.So, this tells that GATA23 is important for defining lateral root primordia specific developmentalprogram.This is just example or the picture which is showing that where it is present exactly.And if you look this is emerged primordia and these are the non-emerged primordia.You can see clearly that in gata23 mutant, the emerged primordial numbers are significantlyreduced and when you overexpress it is getting increased.Same is true for the non-emerged primordia; so, there are a lot of primordia which arebeing generated inside, but they are not emerged.If you count them or or if you go through this and try to count you find that non emergedprimordial number is also decreased when GATA23 is mutated and when GATA23 is induced youcan see that number of primordia significantly induced.So, this all if I summarize in lateral root development.So, what happens that first important thing is that auxin is regulating almost every expectsof lateral root developmental program.And there are different set of modules of auxins regulating different program and thetotal program which as I said that though we see lateral roots in the maturation zone,but the program or the developmental programming has started as early as in the basal meristem.So, this is meristematic zone at very tip of the root, in meristem we have this basalmeristem.So, the priming of lateral root program initiates in the in the basal meristem and here auxinbasically is working through this module auxin Aux/IAA 28 ARF 5, 6, 7, 8 and 19 and thisauxin specific model is responsible for priming the developmental program for lateral root.Then in the later stage this is second auxin signalling module where auxin is working throughAux/IAA 14 and it is regulating ARF 7 and 19 and they are basically helping in the lateralroot founder cell polarization, here is the nuclear migration zone you can clearly see.And then if you come slightly later stage there are two modules which are working inparallel and they are regulating lateral root initiation and patterning development.And in the maturation zone, there is yet another modules of auxin or kind of similar modules,you can say this is module 2 2 dash and these models they are parallel working in regulatinglateral root emergence or the zone where you can start seeing lateral root coming out.So, here we stop lateral root development in next class we will take shoot development.Thank you very much.