Welcome back to Plant Developmental Biology course.So, we are continuing Root Development, in today’s class we will discuss vascular tissuedevelopment.So, basically vascular tissue is very very important and it is it makes basically transportsystem in the plant.And transport system is absolutely essential for survival of a plant.We also have transport system in every organism, in animals, the nature of transport systemsalso called circulatory system it is because there is a circulatory mode of transport wherewe have a central organ called heart.But in case of plants, transport system is not circulatory it is non-circulatory andit is parallel two parallel tissues xylem and phloem’s basically makes the transportsystem.And xylem what it does it takes mostly water, minerals from the soil and it transport everywhere.And phloem basically takes photosynthetic material and lot of signaling molecules andit distributes or allocate to other part of the plants.Another important and key difference between the transport in the transport system betweenplants and animals are, in case of animals transport occurs through the tube which iseither artery or veins.So, basically there is a very specialized structure which is called tube and the transportoccurs outside the cells.But in case of plants basically the transport occurs through the cells.So, there are special cells they have acquired special features and the transport occursfrom one cell to another cells and it is moving for both in case of xylem as well and thephloem.And, basically the vascular tissues have three different components xylem, phloem and cambiumor procambiums.Procambiums are basically kind of stem cells for generating other these secondary tissuesduring the process of secondary growth which makes cambium.But another important thing, that the organization of these tissues xylem and phloem is dependenton the tissue.So, if you look the root there is a different pattern of vascular tissue arrangement, ifyou look shoot there is a different pattern of tissue arrangement leaf and everywhere.And the process or the developmental program for vascular development is set at the stageof embryogenesis itself the primary vascular tissues.So, as you can see here may be in next slide you will see more detail, but these tissuesare basically your procambium tissues; and this procambium tissues are going to basicallymake a kind of cambium cells.This is root tip or primary root tip and if you look the arrangement of this vasculartissue so if you take from the outer side you have epidermis, then you have cortex andthen below cortex you have endodermis and after endodermis you have pericycle.So, basically your vascular tissue, they have pericycles then in pericycles you have thisis a typical primary root of Arabidopsis model plant Arabidopsis.You have xylem and xylem has a kind of linear axis you can see here and in xylem you haveprotoxylem and metaxylem, so these two are the protoxylem they are the metaxylem andthen at the two poles you have phloem tissues.So, these are the phloem tissues pole and in this phloem tissue, there are two veryimportant phloem tissue these are called sieve element and then companion cells these arevery important so sieve elements and companion cells.And if you cut cross section at different places the arrangement or organization ofthese tissues is different.So, the question is that how this vascular tissues are specified, how procambiums orvascular procambiums are specified.So, if you look the embryogenesis early embryogenesis, so basically these are your ground tissueinitials or the putative ground tissue they are going to take and then these are the vascularstem cell initials.And what happens here at this stage this is may be more clear, so these are ground tissueprecursors; and then you have pericycle and then you have vascular stem cells.And again I would a say that auxin is very very important in all these kind of cell specificationand patterning and auxin is basically polar transported by PIN protein.So, this PIN proteins are very very important in distributing auxin basically, helping ina particular tissue or in a particular organ to have auxin maximum.And another important thing for this PIN proteins are that this PIN proteins are asymmetricallylocalized in the cell.So, in one cell wall they are localized and their asymmetric localization basically providesa direction for polar auxin transport because, they are the transport proteins.And if you look at the early stage what happens is that, auxin is polar transported and thispolar transport activates a class of transcription factor this is MP is again a kind of auxinresponse factor and this MP activates a key regulator of a procambium identity or specification,which is ATHB 8, HB8 is again homeobox class of transcription factor.So, this is how the program of pro cambiums specification takes place at embryogenic stages.In later stages in the in the root if you look how the procambium state or stem cellfate is maintained or regulated.So, this is a cross section a typical cross section of root.So, this is your xylem axis, this is the phloem pole and these are the procambium tissues.And what happens that there is a there is a signaling between phloem cell and and procambiumcells.So, from phloem cells there are two signaling two putative signaling molecule CLE41 and44 they are being received by the procambium cells through PXY and TDR based receptionmechanism.And this eventually activate another class of homeobox protein which is called WOX4 andWOX4 basically helps in specifying stem cell fate to the procambium.Then how tissue pattering are happening?So, auxin and another very important hormone is cytokinin, there antagonistic interactionensures a proper tissue arrangement in the root.So, if you look how auxin and cytokinins are distributed in the root these are IAA2 promoterdriving GUS.IAA2 gene is target of auxin signaling pathway.So, which means that wherever this expression is there, it means that those cells have activatedauxin signaling program.And this is ARR5 which is response regulators, and response regulators are basically downstreamgenes regulated by cytokinin signaling pathway.So, if you see expression pattern here, you tells that these are the cells where cytokininsignaling are active.So, if you look here in the primary root what happens that, auxin is very high in the xylemaxis with little in the procambium cells.And in xylem axis protoxylem are having slightly more than the metaxylem it looks like.However, if you see the cytokinin signaling the cytokinin signaling are very less in thein the xylem tissues, but in procambium tissues the cytokinin expressions are high.And this kind of pattern this is a kind of antagonistic pattern, basically defines regulatorymechanism for different tissues.So, what happens in the protoxylem cells what happens you have a very high amount of auxinand auxin basically activate AHP6 protein and this AHP6 protein it goes and inhibitscytokinin signaling in the protoxylem.So, in protoxylem for protoxylem to be as a protoxylem, what has to happen there shouldbe very high auxin signaling and no cytokinin signaling or very low cytokinin signaling.Another thing what happens that this auxin activates TMO5 and LHW and this activatesLOG4; LOG basically a gene which play a role in cytokinin biosynthesis and which resultsin cytokinin biosynthesis.But this happens in the protoxylem, but this cytokinin move to the procambium and in procambiumthis cytokinin signaling is getting activated and this cytokinin signaling in the procambiumcells activate all the genes or all the programs which is required for procambium cell maintenance;whereas, this auxin signaling is working in the xylem axis and regulating the differentiationof xylem tissues.Another regulatory mechanism which works in defining cell fate or identity of xylem cellsis through SHORTROOT and SCARECROW protein and this is very very important.So, if you look here and if you recall your previous class what happens that this SHORTROOTprotein is synthesized in the vascular tissues?So, you have vascular tissues these are the vascular tissues, in vascular tissues youhave synthesis of SHORTROOT protein, but SHORTROOT protein they moves to the endodermis.So, this is your endodermis and in endodermis where SHORTROOT protein moves.So, protein is made in the vascular tissues and it is moving to the endodermis.And in endodermis it get nuclear localized and once the SHORTROOT protein enters insidethe nucleus, it activates SCARECROW; and SCARECROW and SHORTROOT protein they together activatemicro RNA micro RNA 165, 166.And these micro RNA they are basically getting produced in endodermis and then they are diffusingback in the vascular tissue and this diffusion, basically what does it creates a kind of gradient.So, if you look here so the cells which are directly or the cells which are closer tothe endodermis will receive high amount of micro RNA and the cells which are away fromthe endodermis they will receive low amount of micro RNA and that is why you have a gradientof micro RNA higher to lower side; and this gradient of micro RNA is also reflected inthe targets of micro RNA.So, if you look here what happens this is micro RNA.So, micro RNA level is very high in endodermis and then micro RNA level is going down onceyou move through pericycle, protoxylem and in metaxylem.So, in metaxylem what you are expecting very low amount of micro RNA, but in in protoxylemyou are having high amount of micro RNA.But if you look the target, so basically this micro RNA targets homeodomain containing transcriptionfactor PHABULOSA and that kind of transcription factor we have seen in in one of the previousclass.But, what happens that the metaxylem will have high amount of homeodomain proteins andthen the protein levels will go down.So, the cells like pericycle or protoxylem, let us see between protoxylem and metaxylem.So, protoxylem is going to have high amount of micro RNA, but low amount of HD ZIP proteinmetaxylem is going to have low amount of micro RNA and high amount of HD ZIP protein andthe amount of HD ZIP protein basically defines the identity of a cell.If HD ZIP protein is low in amount, the cell is going to be protoxylem protoxylem and ifHD ZIP protein is high in the cells it is going to be metaxylem.So, basically these kind of regulatory mechanisms is defining xylem cell identity.So, apart from these specification and identity, there are another very special feature whichis associated with the xylem cells which is a kind of secondary wall formation.So, if you look normally what happens that if your once your cell is specified as a xylemcells, this xylem cells enters in the process of secondary wall patterning and there arelot of changes occurs and eventually the xylem cells they die through the process of programmedcell death.And they this is very important, because xylem has to work as a tube for conducting waterand other minerals or it has to have a very strong mechanical property to be able to transportthis.So, the this process of xylem differentiation is also regulated by another genes some ofthese genes are VND6 and 7, SND1 and these are LOB domain containing transcription factorand they are eventually regulating some MYB domain transcription factor.And this this interaction of all these proteins basically, they ensures that there are somecellular modification by depositing some cellulose, xylan, lignins.And then eventually some of them are regulating the final process of programmed cell deathto generate a final functional xylem tissues.Now, another important tissue which is part of vascular tissues are phloem tissues ifyou look here.So, this is the picture which is which tells that they are running in the parallel so thisis basically your xylem vessels and this is your phloem tissues.In phloem tissues there are two important tissues one is called phloem sieve tubes orsieve element or sieve cells and this is directly connected with companion cell from one sideand phloem pole pericycle from another side.So, if you make a cross section here you can see that these are the sieve elements, theseare the companion cell and these are the pericycle cells, which is directly attached with thephloem pole pericycle cells it is called.And early stage of these phloem development is regulated by these mechanism this has beenidentified OPS-CLE40 mediated BAM regulation with this is also working in the dose dependentmanner and BRX based mechanism.So, these mechanisms or these regulators together they are functioning to ensure a proper protophloemspecification.Protophloem specification these phloem at very early stage in the meristmetic zone orupto elongation zones are basically called protophloems.These protophloem then undergo the process of special differentiation program and thisdifferentiation program is highly regulated again which we will see later.But another very important regulator which regulates phloem development is another transcriptionfactor which is called APL APL transcription factor.As you can see that and this regulation of phloem is very very important if you do nothave phloem in this apl mutant phloem identity is totally lost and this mutant is seedlinglethal.So, plants are grown, but it cannot survive for the entire life and if you look what kindof phloems are defective in these mutant background.So, these are the markers J0701 basically marks your sieve element or protosieve elements.And if you look in normal wild type root you see this markers are present, but in apl mutantthis markers are absent which tells that the sieve elements or protosieve elements aremissing or there identity is not established.The SUC2 protein and SUC2 is a marker for companion cell and you can look here thateven companion cells are not formed in the apl mutants.So, this tells that APL regulates both the cell types sieve element as well as companioncell identity in in the root.And if you make a cross section this is your wild type you have xylem axis then you havethis phloem cells, but if you look the mutant background this identity of phloem cells aretotally different.But if you complement with the APL you can see again the normal function.Then you know that APL is regulating both companion cell as well as sieve element, butwhere does APL itself express.So, there are two way of regulation one regulation is that which is called cell autonomous regulationor non cell autonomous regulation.So, basically what happens that if the regulator is present in the cell and then regulatingits identity then it is called cell autonomous.Protein is present in one cell and regulating identity of the neighboring cell or some differentcell then it is called non cell autonomous regulation.And when you check the expression pattern of of APL protein it express both in protosieveelements this is basically nuclear localized signal you can see in the sieve element thisis protosieve elements and later stage you can see that this is your protosieve elementand then the companion cell in the differentiation zone these are the companion cell.So, this express both in sieve element as well as companion cells and regulates theiridentity.Then next ok, so this is a regulator of both the cells, do we have some specific regulationof the sieve element cell to identify these there was genomics approaches taken.So, what it has been done, so there was approach called microarray; if you recall we have discussedin some of the previous class and what happens that microarray has been performed, but itwas performed in a very precised manner this phloem cells were sorted using a techniquecalled FACS.FACS is florescent activated cell sorting based techniques.So, if you put some florescent tag in these cells, then specifically you can isolate thecells from these tissues.And, then phloem cells are the cells which are present at the place of phloem has beenisolated from apl mutant and then total RNA was extracted and it was used for doing microarrayand to identify what are the genes whose expressions are getting affected; and out of these genesthere were large number of genes, but two important transcription factors were identified.So, one was NAC45 and another was NAC86 so both are NAC domain containing transcriptionfactor NAC45 and NAC86 and ok.So, basically what happens here, as you can see that in apl mutant basically this is expressionof NAC45, but NAC45 expression totally absent in the apl mutant.Similarly, if you look here this is apl mutant and NAC86 so cells are absent.So, this tells that the both NAC45 and NAC86 are down regulated in apl mutant background.Then if you check the expression of NAC45 and NAC86 itself what you find that thesegenes are very specifically expressed in the sieve element, but they do not express inthe companion cell.So, this tells that they might be they might regulating sieve elements cells not the companioncells and this was true when we make mutant single mutant of nac45 or nac86 not give anyphenotype, but when you made double mutant what you can see that there was a defect here.And then if you analyze this mutants or the wild type pattern of sieve element differentiationin details what happens that, if you look so this is basically tracking one single layerof sieve elements and what you can see this is towards the root tip this is towards theupper side.And at the root tip sides you can see that sieve element cells they are looking verynormal very normal in the meristematic zone and then suddenly what happens that thereis a cell elongation the cells started elongating and then some changes started in the phloemcells; and this changes immediately turns in a way that this cells degrades all thecytoplasm including the nucleus.So, sieve element the functional sieve element is enucleated there is no nucleus most ofthe cytoplasmic components are degraded and this is essential for its function of transportas you know that transport is occurring inside the cell.So, cell has to have minimum inhibitory components here for the transport system and this happensin a very very specific manner.How this is if you look there are three stages first stage it is very normal, second stagewhich is called intermediate the process of differentiation or special differentiationof cell sieve element started and then in stage three the differentiation has occurredin a way that the sieve elements has totally lost the nucleus now it is empty more kindof channels and the common cell wall between two cells are very porous in nature, in caseof sieve element that is why it is called sieve?Because there are holes there are pores in the cell wall and it makes a kind of structurelike sieve and that is important for water or anything to move from one cell to anothercell.If you look the mutant this double mutant what we see that cytoplasm and nucleus degradationis blocked.So, there is no degradation since there is no removal of nucleus there is no removalof cytoplast the sieve elements are not completed its differentiation program and that is whythese plants are also seedling lethal they do not survive for life long and you can checkhere.So, this is longitudinal view of your root tip, you can clearly and very specificallysee the sieve elements of because of it is a very special feature you can see that ithas a very thick cell wall which distinguish from other cells.And if you look this is CAL 7 promoter which is specific for the sieve element and if youdrive H2B is HISTONE2 protein.So, basically this is nuclear localized protein you can clearly see that till here in thesieve element profile there is nucleus, but here onwards this is the sieve element cellswithout nucleus.So, nucleus is totally disappear this is the enlarged view, so if you look here up to herethere is a nucleus, but here you can see there is a diffused GFP signals means nucleus isunder the process of degradation.And then the just next cell here you can clearly see that nucleus is almost removed.But if you look the mutant in mutant what happens that nucleus is not degraded evenif you go to the very higher side of the cells where cell, sieve element cells are very verylong, but you can still see the nucleus.This suggest that in the double mutants nucleus is not degraded and then there is a mechanismwhat is the mechanism exactly which is regulating the process of nucleus degradation this wasagain to identify.So, NAC45 and NAC86 is regulating nucleus degradation, but it is transcription factorit cannot go and directly regulate the process of nucleus degradation, it must be regulatingsome of the genes which is directly involved in the process of nuclear degradation.To identify these genes another round of microarray was performed using this double mutant andNAC45 overexpression.And from this microarray analysis there were four genes very small family of putative nucleasefamily of genes we are identified which was NEN1, NEN2, NEN4, 3 we are showing here.And what you can see there is a very important thing what is happening first thing that thesegenes are expressed in the sieve element which is basically correlating with the fact.And, another important thing if you look the sieve element at early sieve elements or youngsieve elements the gene is expressed in the sieve element protein is made, but proteinis not able to enter inside the nucleus this is the nucleus and protein is entering onlyin the cell where nucleus has to be degraded.So, before this cells protein is not able to enter inside the nucleus so there are tworegulation one regulation is that this nucleus genes are getting expressed in the sieve elementthat is regulated at the transcription level and then another regulation is at the posttranslational level when protein is already made its translocation to the nucleus is regulated.Similar kind of things you can see for NEN2 as well where as if you will see NEN4 thisis this is specifically expressed only in the cell where nucleus has to be degraded.So, if you look early cells does not express later cells nucleus is degraded.So, there is no nucleus no signal, but this cells has NEN4 protein localization only insidethe nucleus.So, all this story tells that this putative nucleases might be responsible for nucleusdegradation.And this is supported genetically when you have nen4 mutant background you can see thatnucleus degradation is defective in this mutant background.So, here everything is normal except NEN4 is not present.So, if you take all these together we suggest that APL is regulating both sieve elementas well as companion cell.And then, during this process APL is regulating NEN NAC45 and 86, NAC45 is regulating sieveelement differentiation, but cytoplasm as well as nuclear degradation, but it is regulatingNEN1 to 4 and this NEN is specifically regulating nucleus degradation.So, this is how the process of phloem specification takes place to make a mature phloems maturesieve element cells.And as I say this mature sieve element cells is very special it has this sieve pore likestructure this is almost empty, so it is very suitable for very high or efficienttransportsystem.So, in general to summarize the vascular development, so what we have studied.There is auxin auxin is regulating auxin response factor MP and it is regulating ATHB4 and thisprocess is basically ensuring vascular stem cells through the WOX4 genes.There is another regulation from the phloem sides which is CLE 1 CLE 41 and CLE 44 mediatedregulation and it is working through a PXY/ TDR to regulate this stem cells identity.And then later on what is happening there is a mechanism through SHORTROOT protein andSCARECROW protein micro RNA and HD ZIP mediated protein, this is basically regulating xylemcell identity protoxylem versus metaxylem identity.Another regulators which is VND7 which is regulating again protoxylem identity VND7regulating metaxylem identity.But on the other hand if you look APL genes APL is basically activating phloem kind ofidentity, APL is regulating companion cell and sieve element both and for sieve elementbased specification it is using NAC 45, NAC 86 and NEN mediated signaling pathway.And then protoxylem is also regulated by the feedback of auxin, AHP6 and cytokinin basedmechanism.So, will stop here in class we will discuss root branching.Thank you very much.