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Welcome back to Plant Developmental Biology course.So, we are studying today Shoot Development particularly Organogenesis part.So, in the last class if I recap we have studied shoot apical meristem or maintenance and herein this class we are going to study organogenesis.So, if you recalls this is typical shoot system in plant and in shoot system we have basicallyfew organs like during vegetative phase we have leaves and axillary branches and at theapex we have shoot apical meristem.But, when the transition occurs from vegetative phase to reproductive phase, this shoot apexshoot apical meristem get converted into inflorescence meristem and then inflorescence meristem makesthe flower.So, these are the typically part of shoot system.And you have seen in previous classes that how shoot apical meristems are organized duringvegetative growth and development.This is just to recap that this is a vegetative shoot apical meristem and if you rememberthere is a central zone and then you have rib zone and you have peripheral zone, andthis is the top view of a growing shoot apical meristem.So, the central region is shoot apical meristem, but if you look in the peripheral zone orperipheral region of the shoot apical meristem, these are the organs which are under the processof development.So, one important thing what happens during the growth and development is that, whilethe meristematic activity is maintained in the very tip of the meristem, when the cellswhich are produced in the meristem, when they enters in the peripheral zones they basicallyinitiate the process of differentiation.So, here is the view of inflorescence meristem so, it looks very similar.So, in inflorescence meristem also you have a central zone then you have peripheral zonesand rib zones and stem cells are maintained in the central zone.So, this is the region where cell division activity is very dominant and differentiationis inhibited.But when this cells enters in the peripheral zones they basically initiate organ specificdevelopmental program depending on the fact whether if they are in the vegetative phase,the lateral organs are going to be the leaf if they are in the reproductive phase, thelateral organs are going to be a flower.So, one important thing what I would like to tell here that, this both the process,process of meristem maintenance and the process of organogenesis is highly coordinated andit occur simultaneously.If this does not happens, if let us assume that meristematic activity is more than thedifferentiation activity then the meristem size will keep on increasing.On the other hand if you have more differentiation activity then meristems will be consumed early;under both the conditions you are going to see abnormal growth and development.So, during the process; both the process has to be coordinating.So, in last class we have seen how meristems are getting regulated, what are the geneticregulatory pathways particularly WUSCHEL and CLAVATA mediated pathway and how they basicallyensures meristematic activity in the meristematic zone.Here in this class we are going to see how the differentiations are initiated.So, what happens in the differentiation so if you look this is a central meristem andin the peripheral meristem, first side where the process initiate is called I1, I2 likethat and then you have a leaf primordia like P1, P2, P3, P4, P5, P6 and so on.So, basically if you look there is a clear defined pattern of organogenesis.So, organogenesis is not a very random process if you look the top view of a growing meristemthat between P1 and P2 there is a typical angle.So, basically the positioning the primordia is another very important and critical parameterduring the process of organogenesis.So, everything is being maintained here and how these primordia’s are positioned, howit is being define then there was a hypothesis that some kind of inhibitory zones are createdaround a primordia.So, if you look if these are the primordia, the circles basically denotes the inhibitoryzones, which means that in this region or around this primordia up to this region thereis no other primordia will originates, but question is still here that how and so preciselythis inhibitory domains are maintained.Another thing what is important which I was telling that a coordination and communicationbetween meristem and growing primordia is very important.How will you prove this?So, for example, if you look this and if you kind of create a kind of incision betweengrowing primordia and the meristem.Basically if you physically disturb or you are removing one of the developing primordia,what you are getting here that the patterning or phylotaxis is disturbed.For example, if you make incision between P1 and this center of a meristem what yousee that the positioning of I2 is now changed.So, it is basically shifted from the original position.Similarly, if you remove or somehow kill the primordia one of the primordia if you lookthat there is a migration or there is a change in the position ofother primordia.So, this tells that some kind of communication between meristem and the primordia is goingon and this is absolutely important for positioning primordia in the growing meristem.What could be that signal or what could be that communication way?So, one thing which is clear and you would have seen in previous classes as well thatauxin hormone which functions like a morphogen is absolutely important for entire growthand development.And if you look the DR5 activity which tells the auxin response activity if you look hereso, in the primordia in the growing primordia auxin amount is very very high and then ifyou look some of the mutants.So, if you look the pin mutant; PIN is basically a protein which is responsible for polar transportof the auxin.How it happens, I will just discuss here, but if you look the pin mutants if you donot have PIN protein which means that the auxin is not getting distributed properlyyou look the structure that organogenesis is almost blocked and the structure lookslike a pin.Whereas, if you just apply little bit of auxin exogenously micro application of auxin youcan see that organogenesis can initiates.So, this suggest that auxin is playing very very important role in the organogenesis orin the primordia differentiation.How the auxin basically works?So, auxin is bio synthesized in some of the cells and then they can be distributed orthey can be transported and the transport of auxins occurs through a well establishedauxin transport system which is also called polar auxin transport system.Because the transport of auxin is a directional and how it occurs?If you look the IAA; IAA is one auxin when in the cell wall and the cell.So, basically there are two kinds of protein AUX protein and PIN protein.So, PIN proteins are auxin efflux carrier AUX protein are auxin influx carrier.So, through these transport systems auxins are being transported from one cell to anothercells.Another very important thing which is always associated with the PIN proteins are thatthey can be asymmetrically localized in the cell.So, one particular cell wall may have the PIN proteins, which means that they are asymmetriclocalization in the cell wall can provide a direction for auxin movement.So, if PIN is localized here then auxin can move from here to here, and this kind of polarauxin transport basically helps in gaining auxin maxima in some of the tissues.So, one hypothesis was that there could be that auxin is getting accumulated very highin the primordia and this is happening through the polar auxin transport.And this has been tested and this has been shown in a great detail using different mutantsor different approaches that what happens.If this is a meristem then auxins are getting transported towards the position where a primordiahas to be initiated.And this is how the region or the tissues they basically achieve auxin maxima, and whenauxin level is increased beyond a certain point it initiate a primordia specific developmentalprogram.And then another thing is important that it can initiate program then the auxin can bediverted from the place.So, that a crucial balance is always or homeostasis of auxin is maintained.So, if you look the central zone, in central zone a cytokinin is predominant and then youhave a KNOX gene which is responsible for the maintenance, but in the primordia KNOXgene expression is low cytokinin is low, but auxin is high.So, the critical balance between auxin and cytokinin and some of the key transcriptionfactors defines the meristematic activity in the meristem and differentiation activityin the primordia.This you can also see by the genetic interaction.So, this is a double marker here you have DR5 is auxin responsive promoter driving anuclear localized yellow signal which is basically auxin response and PIN protein has a GFP.So, you can see the GFP localization as well as the auxin response and you can see thatauxin response are very high in the primordia.And MP is a transcription factor which is a auxin response transcription factor whichis regulated by the activity of; MP is regulated by auxin and what happens if you have singlemutant of mp you can see that the differentiation or the phyllotaxis or the organogenesis isdisturbed.But if you combined mp with the pin mutants, were basically you have the response auxinresponse factor mutant as well as the polar auxin transport mutants you can see the phenotypewhere organogenesis is almost blocked.If I summarize here you have auxin and then auxin is getting diverted in the site of primordiathrough the polar auxin transport.And then in the primordia you are going to have a very high amount of auxin and thisauxin initiate the program and then auxin has been diverted from there through the vasculartissues.So, this is what happens in the meristem; growing meristem.So, you have a central meristematic zone and you have a peripheral primordia and the anothervery important thing is the border or the boundary between meristem and primordia thishas to be defined.So, up to what region the cell division activity should occur and then when the differentiationinitiate and this kind of boundaries are maintained by some of the genes which are very specificallyexpressed in the in the boundary for example, CUC2 they are LOB domain containing some ofthem are NAC domain containing and some of them are LOB; LOB is lateral organ boundarydomain containing transcription factor.So, if you look here in the meristem you have WUSCHEL and CLAVATA activity, high cytokininWUSCHEL and CLAVATA activity and the gene meristem regulating gene like STM they ensuresmeristematic activity here, but if you look in the primordia you have high amount of auxin.Auxin is activating, AUXIN RESPONSE FACTOR5 or MP and then they are activating some leafprimordia or floral primordia a specific genes which are basically regulating primordia differentiation.The coordination between meristem and the lateral organs is well established.And if you look this there is a feedback from lateral organs which also controls the shootapical meristem activity.So, it is signaling both way.So, meristem is controlling the activity of lateral organ growth and lateral organ isgoing to control the meristematic activity, how?If you look here PIN protein localization in the wild type this is primordia P1, I1,I2, if you look the PIN protein; PIN protein is mostly localize to the L1 layer.Whereas, if when this is a normal growing primordia, but if you remove one lateral primordiawhich could be leaf primordia if you just remove it what is happening that distributionor localization of PIN protein is totally disturbed here.So, this tells that the presence of lateral organ or development of lateral organ in theperipheral zone is sending some kind of signal to the meristem for its own activity.And if you produce auxin in high amount so, basically in the CLAVATA3 promoter you areusing CLAVATA3; promoter CLAVATA3 is expressed in the central region and if you synthesizeauxin what you see that the meristematic zone is decrease the meristem size is decreased.On the other hand if you remove auxin from the meristem.So, basically this YUCCA genes are auxin biosynthesis genes.So, if you have double mutant of YUCCA1 and 4 or triple or four mutants of different membersof YUCCA gene family.So, essentially what you are doing?You are reducing total bio synthesis bio synthesis of auxin and in that case what you can seethat the meristem size is increasing.So, this tells that there is a clear feedback signaling between lateral organs as well asthe meristem.So, under normal condition when your shoot apex is here, you have lateral organs coming,then this is your central zone auxins are getting transported to the primordia and thenin primordia it is initiating program whereas, there is a feedback regulation here.But, if you remove organs the programming is totally disturbed as you can see from thePIN protein, the distribution of auxin is not happening in the normal way this is affectingmeristematic activity as well as organogenesis.Another important thing, so apart from that the organ specification or organ identityor differentiation one more thing which is getting ensured at this early stages organpolarity.So, if you look any lateral organs whether it is flower or in different organs of theflowers sepal, petals, stamen or carpels or if you look the leaf they display a kind ofpolarity.So, if you look leave.So, here is the polarity if you look their basal side of the leaf which is very differentthan the apical side.So, here if you look a typical leaf so, the basal side of the leaf and apical sides ofleaves are very different.So, this kind of polarity is called proximal and distal polarity.On the other hand if you look the leaf so, the leaf which is towards the meristem isa kind of adaxial and the portion which is away from the meristem is called abaxial.And if you look that there is a clear organ polarity, and this polarity is also need tobe established at the time of organogenesis.So, for example, if you look a schematic diagram what happens in the growing meristem?So, this is meristem and the this is the lateral organ and in lateral organ the face of thelateral organ which is towards the meristem, this is called adaxial.And the part of them on the lateral organ which is away from the meristem is calledabaxial, and if you look the features of adaxial and abaxial surface, it is very different.So, that is and this has to be established early.And in the leaf, if you look the leaf growing primordia.So, this is your adaxial side this is your abaxial side and if you look the positioningof the vascular tissue.So, xylem is positioned towards the adaxial sides phloem is positioned towards the abaxialside and there are lot of genes which is responsible for maintaining this polarity adaxial versusabaxial polarity.For example, if you look some of the mutants if you have this mutants you can see thatthere is a radial pattern and you can see that xylem is in the center and phloem isthe peripheral.If you look phabulosa mutant here it is opposite pattern you have a phloem in the center xylemin the so.So, in both the cases what is happening that polarity is disturbed.So, this polarity establishment occurs very early when meristem is getting maintainedin the center and primordia and the lateral organ primordia is initiated.And the positional single plays very important role and this is clear from this experiment.So, what happens that if you take this is a growing meristem and then you have a lateralprimordia.So, this what I said that this is your proximal region, this is distal region, this is youradaxial region, this is your abaxial region and what you do?If you just make a very small incision basically in just the L1 layer between primordia andthe meristem if you make a kind of incision.Here in L1 layer, what you see that the primordia; the leaf primordia which is coming here ithas totally lost the polarity.So, it looks like a very different or more kind of radial patterning whereas, if youlook this primordia which is coming from this side, it is still maintaining the polarity.You can clearly see the adaxial side is very different than the abaxial side whereas, hereeverything is looking like radicalradial pattern.And this suggest basically that there is a communication between meristem and primordiathat is also important for establishing organ polarity here.So, if you look this schematic diagram, this is the top view you have a central zone andsome kind of signals are coming from here and these signals are helping and definingadaxial versus abaxial polarity in the lateral organs.There are some of the genes which has been identified to regulate this organ polaritywe will discuss in the next class.So, some genes are regulating adaxial surface some genes are regulating abaxial polaritywe will see.Another important thing here is that the signals derived from YABBY gene activity in organprimordia regulates growth and partitioning of Arabidopsis shoot apical meristem.So, it is not only that the meristem is regulating the organ, but the organ specific genes arealso regulating the meristematic activity.So, YABBY class of genes are the genes which regulates which provides abaxial polarityin the organ.You will see in next class in detail, but what happens here that if you look the expressionpattern of FIL which is a kind of YABBY gene, what you see that this is your inflorescencemeristem expression in the inflorescence meristem per say is very very low or almost not detected.But if you look the primordia, lateral organ primordia expression is very high, but expressionis more towards the abaxial surface then the adaxial surface.If you look the top view of it you can clearly see that this is the primordia, but in theprimordia expression is only restricted away from the meristem which is your abaxial surface.So, this suggest that these genes might be regulating abaxial fate.And, what happens in this mutant background if you look this is a filamentous and thedouble mutant filamentous and yabby3 mutants, what you see that the defect in the polaritygenes in the organ polarity genes is basically disturbing the phyllotaxis.The arrangement of the organs which is a feature of ahh meristematic activity.So, this is visible from here if you look, the distance between two, primordia two adjacentprimordia is here in the wild type, but in the mutants this distance is basically changed.So, this suggest that the polarity genes which are expressed in the organ they are also regulatingthe meristematic function in the shoot apical meristem.Some of the markers so, two very important and well established meristematic markersare WUSCHEL and CLAVATA.So, if you look the CLAVATA expression domain.So, in wild type this is the CLAVATA expression domain and this is in the very tip restrictedhere in the center, but if you look the single mutant the domain of CLAVATA3 expression isexpanded, and double mutant it can be even further expanded.Similarly if you look WUSCHEL expression domain here in the wild type it is restricted veryin the few cells here.But if you look the single mutants or double mutants the WUSCHEL expression domain is significantlyexpanded both laterally as well as in the apical and basal domains.So, this suggest that the meristematic activity or the regulators of meristematic activitiestheir expression pattern and their localization is disturbed when you have defect in the organpolarity.So, in general if you look here.So, there is a meristematic activity some of the genes which is regulating meristematicactivity, and then in the peripheral zone the founder cells has been specified whereauxin and some of the regulators are playing very important role and then outgrowth andpolarity is regulated.So, these are the three different steps during the process of organogenesis.So, there are some genes for example, ASL, LEAFY, ANTIGUMETA, CUC, KANADI all these genesare basically regulating the process together.So, if I summarize here.So, you can see that in the meristematic zone, where you can see CLAVATA and WUSCHEL mediatedsignaling and cytokinin these are the key, but there are many other genes as well theyare regulating the meristematic activity.But in the peripheral zone in the primordia you have high amount of auxin; auxin is regulatingsome of the transcription factor and this transcription factors are basically activating,organ specific genes LEAFY, ANTIGUMETA, like genes and these genes are ensuring a properdifferentiation.At the same time what is happening that, there are some signals, which is derived from themeristem.And they basically is activating some of the gene like PHABULOSA in the adaxial side ofthe lateral organs.And some other genes are getting activated in the abaxial side of the lateral organsand then there is a kind of crosstalk and regulation between adaxial and abaxial genes.And this regulation is basically ensuring a proper polarity in the growing lateral organs.So, I will stop here in next class we will discuss leaf development.Thank you.