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Welcome back to Plant Developmental Biology.We are continuing Shoot Development and particularly organogenesis during shoot development.So, in previous class we have discussed the coordination between shoot apical meristemmaintenance as well as the organogenesis in the peripheral region.In this class we are going to take example of leaf development, you know that duringvegetative growth the major organs which a plants make is leaves and there is a hugevariations in the morphology of the leaves across the plant.So, you can have different plants having a different shape, different size, differentarrangements of the leaves.So, for example, if you look maize, Arabidopsis, rose, there is a huge variety in the morphologyof leaves.But if you look the pattern of their development, so there is a quite similarity of course,there must be some species specific differences.But what happens generally during the leaf development, as you have seen in the previousclass that the meristem is being maintained here in the center.And then in the peripheral region the primordia is getting positioned.So in case of leaf development the primordia is going to be leaf primordia and this ispositioned by generating auxin maxima in the peripheral region.And the one auxin maxima is generated here, the next auxin maxima will be generated ata particular distance from the existing leaf primordia.And then like that you see early primordia patterning and differentiation.So, once the primordia is positioned then primordia will start the program, which isleaf specific program and then during the differentiation it will make all the tissues,which are specific for the leaf, it will ensure the polarity in the leaf which is adaxialand abaxial and the proximal and distal polarity.So, how it happens this is a kind of typical picture of tomato shoot you can see that thisis meristem this is the leaf primordia 1, P2, P3 and then if you see the region theseare the morphogenesis.These regions are basically differentiating and eventually they are giving mature leafwhere you have this different part of the leaf.This is a leaf morphology in model dicot plant Arabidopsis.So, Arabidopsis during early stage of the development they have cotyledon type of leaf,which are very small during vegetative growth they makes rosette leaves.Rosette leaves even you can see a variation in the shape size and morphology of the rosetteleaf.And then you have a cauline leaves here these kind of leaves are usually produced in theinflorescence stem after transition.Typical morphology of a leaf, so, in leaf you have petiole here which is called leafpetiole and this region is called leaf blade.Then as I said that you will have midrib here and then adaxial, as well as abaxial sides.On one sides you can clearly see that this kind of trichomes are developed and then youhave stomata formation and this is a cross section of the leaf if you look.So, you have adaxial side then you have palisade cell then you have in between the vasculartissues spongy cells and then you have the abaxial sides and during the development whathappens that, the primordia or the program which is specific for the leaf developmentis getting positioned at the site of the leaf formation.And then during the differentiation the these tissues are patterning these are getting patterned,they are getting positions they are taking a special identity and then eventually thegrowth a leaf is basically restricted.So, these are the some genes, which is known during leaf development.So, WUSCHEL and CLAVATA3 they are more kind of responsible for meristem maintenance thenyou have CUC2.CUC2 is as I said that CUC1 and CUC2, they are the boundary genes they express very specifically,at the boundary of shoot apical meristem and leaf primordia.So, they are basically maintaining the boundary then, you have STM and KNOX gene which isregulating the meristematic activity.You have some other genes like ASYMMETRIC1 which is regulating the leaf patterning.And then you have YABBY, FILAMENTOUS and KANADY as a genes which is regulating abaxial polarityPHABULOSA and other FANTASTICA is a gene which is regulating adaxial polarity.So, the these genes are getting expressed and then they are regulating the function.Another important thing is that you have plenty of mutation, so you have a genes which isregulating leaf morphology.And if you have mutation in that you are going to see the defect in the leaf morphology.For example, if you look this mutant which is serrate mutant, so wild type this is themorphology of the leaf you have petiole, then you have the blade.But if you look here there is a serration there is a kind of change in the morphologyof the leaf.Similarly, if you look asymmetric2 mutants you have a kind of similar kind of phenotype,but phenotypic severity is not very high.But if you make a double mutant between as2 and serrate or as1 and serrate you can seethe phenotype is severe.Similarly, if you take some of this KNOX gene and if you ectopically over expressed youcan see that leaf morphology is totally disturbed.Another things what defines the leaf shape and size is basically curvature or leaf curvatureis one important parameter of the leaf.So, basically how curvature is maintained, so if you have a cell this is a kind of example,take a cell and if the growth occurs uniformly then you have a cell growth in a more kindof spherical manner.But if what happens that if growth dominates if the growth in the central region is more,than the peripheral region then what you have you get a kind of positive curvature and youcan see a structure like this, a curve like this.On the other hand if growth is more in the peripheral region, then the central regionthen you will have a kind of negative curvature and then you can have this structure.A mutate a mutants of Antirrhinum CINCINNATA genes has been identified CINCINNATA geneis a kind of class of transcription factor of TCP family and what it was found that ifyou look this mutant the morphology of leaf is extremely defective.So, this is a typical Antirrhinum leaf you have a petiole and then you have the structureand there is a proper shape.But in CINCINNATA mutants what you see that the curvature is totally disturbed in thismutant background.If you look the leaf arrangement here, if you look the defect in the leaf.So, you can clearly see that may be at the early stage difference is not very prominent,but at the later stage if you look this leaf if you spread this curved leaf you see thiskind of structure.If you look the margin of this leaf and if you see the cellular feature of this leafyou can clearly see that in wild type leaf and the cincinnata mutant’s leaf, has verydifferent feature of the margin cells.Another genes which are known to regulate leaf morphology is AINTEGUMETA.If you look here this is a typical wild type Arabidopsis root, but if you have loss offunction of ANT you can see that size as well as shape of shape is somewhat similar, butsize of this leaf is totally disturbed.And if you look this is a wild type pattern if you have loss of function of ANT, you seethis defect.If you have gain of function of ANT if you have over expressed you can see the oppositeeffect here.So, this suggests that ANT is also regulating leaf morphology.So, in general if you look the development of leave so what happen in the first stepin the shoot apical meristem or at the peripheral region of the shoot apical meristem the firstleaf primordia is positioned.And then this leaf primordia start distal growth which occurs in this direction thisgenerates a kind of two end one is the proximal and the distal region.And then at third stage the blade initiates where what happens the marginal region orthis region is basically getting developed into the blades and the basal reasons arebasically developed into the petiole.Later on there is a intercalary growth it start in the blade region and that basicallyhelps in the leaf blade to take a proper structure.And this is the same schematic diagram or picture where you can clearly see developingprimordia, this is early primordia, this is slightly later primordia this is the leafprimordia which is at this stage of coming out.And there are some genes which has been identified to regulate the process.So, in the meristem you know the meristematic genes are there which regulate the meristemfunction.But in the primordia you have other genes which are getting activated one is that auxinlevel and then auxin what happens that it inhibits the meristematic gene.So, basically in the primordia region meristematic genes need to be suppressed the genes whichare basically negatively regulating the differentiation program need to be suppressed.At the same time the genes which are positively regulating the leaf specific developmentalprogram they need to be activated.So, this is what happens here and another thing what I mentioned is that LOB kind ofgenes are the genes which are present at the boundary between meristem and the leaf primordia;and they are very very important to defining the zone or the specially positioning themeristem or separating meristem with the lateral organ primordia.At the same time you have a set of genes, which are getting activated in the developingleaf primordia and these genes are important for regulating leaf specific differentiationprogram.So, after this there is a process of differentiation, it can be either basipetal, acropetal or bidirectionalwhich means that differentiation can start from here and proceed in this direction orit can start from the basal region and proceeds in the upper direction or it can starts fromthe both direction.And then it generates a kind of gradient, but once this gradient is totally so whenboth the ends are completed then you have no gradient, when there is no gradient essentiallyyou can say that here the differentiation or growth of the leaf stops.So, if you look Arabidopsis thaliana so in Arabidopsis thaliana it occurs in the basipetalmanner.So, if you look this is Arabidopsis leaf at different days this is day 4, day 8, day 12and day 60 and this is a CYCLIN promoter driving GUS.So, CYCLIN is a gene which is which basically marks the dividing cell it is a cell cyclegene, so it is marks the dividing cell.So, if you look at the very early leaf which is around 4 day old, you can see the divisionoccurring almost entire region of the leaf.So, this suggest that region is the differentiation is only restricted at the very tip of theleaf.Whereas, most of the leaf they are having cell division or proliferation activity goingon.But when this leaf becomes older, so if you look in the 8 day old leaf what happens thatthe differentiation signal is migrating towards the down side.So, if you have now around 50 percent of the tip region they do not have cyclin activity.So, they are not; they are not showing the division activity, so which means that thedivision activity is mostly restricted to the basal half region of the leaf whereas,the apical half region or the distal half region of the leaf basically they lack thedivision activity, which means that they are; they are; they are undergoing the processof differentiation.Then if you see at more later stage like 12 days the division activity is very restrictedonly in the very basal region of the leaf; whereas, most of the leaf distal region ofthe leafs are showing pattern of differentiation.Slightly more later if you look 16 days old plant the entire leaf they lack the cell divisionor cell growth activity; which suggest that now this leaf is fully mature it is fullydifferentiated now it will stop the property of differentiation.And then some of the genes which has been identified which regulates this property.So, if you look if you divide this leaf so there is a apical and basal or distal andproximal growth of the leaf then you have this marginal growth.And you can see that some of the micro RNA some of the growth regulating factors GRFor the growth regulating factors and then you have CINCINNATA, TCP genes, the activityof these genes basically ensures proper shape, proper size, proper tissue patterning duringleaf development.On the other hand if you look the activity of YABBY, homeodomain transcription factorKANADI genes, they basically ensures the adaxial versus abaxial property or polarity of theleaf.Auxin is also known to regulate basically leaf flattening in Arabidopsis so the auxinbasically activates kind of a regulates activity of AUXIN RESPONSE FACTORS.So, these are AUXIN RESPONSE FACTOR2 to 4.Then you have MP which is again AUXIN RESPONSE FACTOR 5, then some of the WOX genes and theiractivity together basically ensures not only the leaf differentiation or leaf initiation,but also it regulates leaf flattening in these direction.Now, we will look the leaf polarity.So, as I said that leaf polarity is also very important and because some of the featureswhich are present in the adaxial surface is not present on the abaxial surface and thesepolarity is very very important.And polarity is regulated by a clear two category of the genes.So, one category of the genes which are specifically expressed at the adaxial side of the lateralorgans, another category is expressed specifically on the abaxial side of the development.So, for example, if you look this growing pattern and this is messenger RNA distributionof FIL gene which is abaxial gene.So, if you look the pattern.So, this is developing leaf you can clearly see that expression is mostly restricted theabaxial half of the leaf whereas, the adaxial half portion of the leaf do not have thisexpression.And this expression pattern is established very early even at the primordia stage, sothis is your meristem and this is your first primordia here coming.So, if you look if this is the primordia this region total is the primordia, but expressionis only restricted to the abaxial half of the primordia, but half adaxial primordiatotally does not express this abaxial genes.Whereas, on the other hand or in contrast to this if you look the adaxial genes, whichis like REVOLUTA or PHABULOSA you can see it is showing a very opposite pattern.So, if this is a growing leaf primordia, you can see that the half primordia which is towardsthe adaxial surface is expressed these genes very high.But the abaxial side of the primordia it does not have the expression.So, the asymmetric expression pattern or transcript distribution or transcript expression of thesegenes essentially, provides a proper polarity in the developing leaf.Apart from the homeodomain containing transcription factor in abaxial you have YABBY domain transcriptionfactor and KANADI some of the other genes and what happens in this mutant background.So, if you have a mutant for example, if you have a mutant in revoluta what you can seethat loss of function phenotype is totally where adaxial surface is totally abaxialized.So, you can see some of the picture here, so this is wild type Antirrhinum leaves, butif you have a fantastic a mutant FANTASTIC is a regulator of adaxial surface.So, if you do not have this gene expressed you can see that these polarity in the leavesare totally lost so adaxial surface and abaxial surface both looks quite similar.And if you make a cross section so as I say this is a leaf cross section.So, this is your adaxial side, this is abaxial sides and you can see that xylems are usuallyposition towards the adaxial sides phloems are position towards the abaxial sides.But if you take the mutant and make a cross section it look so it looks more kind of radialpattern.And here you are look that phloem is basically everywhere and xylem is in the center.This suggest that when you do not have adaxial specific genes the all tissues are gettingabaxialized, so you have abaxial type of pattern in the leaf.On the other hand if you have PHABULOSA dominant looking so, PHABULOSA is again regulator ofadaxial surface it is it provides adaxial surface.But here it is a gain of function mutant so, if you have gain of function mutants thenwhat happens that the all the regions are getting adaxialized.So, basically even abaxial regions get converted into adaxial region and if you look here,the vascular pattern here the vascular pattern is very different it is opposite of the lossof function here.So, you can see here xylem is surrounding the phloem which means that xylem is basicallyadaxial side.So, in this mutant background you can see that leaf is mostly adaxialized whereas, inloss of function the leaf is mostly abaxialized.If you look the cellular feature of the adaxial versus abaxial surface.So, if you look scanning electron micrograph picture here.So, in wild type adaxial surface looks like this, abaxial looks like.So, in adaxial surface you can sees that there is a bulges of the cells you can clearly seeit is whereas, if you look the abaxial surface it is relativity smooth.So, bulges are not very very strong, but if you over express ectopic over express FILAMENTOUSor YABBY gene.So, remember FILAMENTOUS and YABBY genes are regulator of abaxial surface.So, they basically promote abaxial surface what happens and if you look their adaxialsurface.So, adaxial surface normally in wild type looks like this you have lot of bulges, butwhen you over express or you put FILAMENTOUS or YABBY3, they becomes more similar to theabaxial surface.So, you can see they are becoming more kind of smooth wise.So, this suggest that when you have a gain of function activity of YABBY3 or FILAMENTOUSbasically they are converting adaxial identity to abaxial identity, but if you look the lossof function.So, if you have a double mutant of FIL and YABBY3.So, basically here both the abaxial promoting genes are lacking in that case if you lookthe abaxial surface even abaxial surface look very similar to the adaxial surface.So, basically this is important.So, you have clear two category of genes the adaxial promoting genes and abaxial promotinggenes if you do not have adaxial promoting genes adaxial sides get converted into abaxialsides, if you do not have a abaxial promoting gene abaxial surface get converted into adaxialsurface.And they are actually regulating their activity as well.So, if you look here, so this is the expression of FIL which is abaxial gene and it is positionedit is mostly expressed in the abaxial side of the leaf and it is absent in the adaxialside.But if you have gain of function mutant of phabulosa, basically what happens the gainof function mutant of PHABULOSA means every side is adaxial sides and this if you lookthe expression pattern of now FILAMENTOUS gene because FILAMENTOUS is abaxial.Now, in this mutants you have a everything adaxial, then you this you can clearly seethat if you have a homozygous gain of function mutants of PHABULOSA 1d you can see the expressionis almost disappeared.So, there is no expression of abaxial specific genes.On the other hand if you look here in some of the cases the expression is there, butit is very restricted it is not the expanded.So, basically its domain of expression is highly restricted and if you look some ofthe organs which are basically adaxialize you can clearly see that their expressionis very very low and extremely disturbed expression pattern.On the other hand, if you look the expression pattern of adaxial promoting gene in the abaxialmutant.So, KANADI genes are basically responsible for promoting abaxial side.Now if you have double mutant here and if you look now REVOLUTA PHAVOLUTA these twoof genes are adaxial side genes you can clearly see in mutants background their pattern istotally disturbed.So, if you look in the wild type they are clearly localized towards the adaxial surface,but if you look the mutant here you can see they are quite uniform or you can see evenexpression in the abaxial surface.And similarly if you have kanadi mutants the abaxial genes they have lost their expressionor they are very very low level expression.So, if we summarize if you look first the growing leaf you have the meristematic zoneand then you have the primordia and then in this primordia, the adaxial versus abaxialpolarity is already established.And in adaxial side what happens that these homeodomain containing transcription factor,which is PHABOLUTA, REVOLUTE, PHABOLUSA they are basically active in the adaxial surface.On the other hand KANADI, YABBY these genes are active on the abaxial surface and theymutually inhibit each others in their domain.So, in adaxial domain these genes inhibit expression of YABBY, in abaxial domain thesegenes inhibit expression of homeodomain gene.And these kind of mutually exclusive expression pattern of adaxial promoting genes and abaxialpromoting genes basically establish the adaxial versus abaxial polarity.On the other hand there are some of the micro RNAs, trans-siRNAs they are also playing avery important role in establishing and regulating, polarity in these organs.If you look the cross section of the leaf, so you can clear see that you can have a twodomain; the adaxial domain and the abaxial domain.So, these are the adaxial domain in adaxial domain adaxial genes like these they are basicallyactive and apart from the adaxial genes you can see that genes which are responsible forthe promoting leaf identity they are also getting expressed.On the abaxial side you can have some of the abaxial promoting genes, which are gettingactive; apart from that you have the activity of auxin, micro RNA, AUXIN RESPONSE FACTORS,MP and some of the other key regulators of leaf differentiation.So, all together activity of hormones activity of transcription factors the coordinationbetween meristematic activity and the leaf primordia set complex genetic program whichis regulating proper patterning, proper differentiation and proper organ development in plants.So, I will stop here in next class we will look floral transition and flower development.Thank you very much.