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Approaches to Plant Development Studies

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Welcome to today’s class of Plant Developmental Biology, where we are going to study how tostudy plant developmental biology.So, one of the very important and and prominent approach which we take to study plant developmentalbiology is Molecular Genetics based approach.So, molecular genetics is basically to study how a particular gene in a genome regulatesa particular developmental process in the plants.So, there are two major approaches in the molecular genetics, which we usually to taketo study plant developmental biology, one is the forward genetics based approaches andthe reverse genetics based approaches.So, in forward genetics what we do we begin with a mutant or with the phenotype, and thentry to identify what is a gene associated with that phenotype.Whereas, in case of reverse genetics first we define our gene of interest, and then wetry to understand or we design experiment to pinpoint what is a specific developmentalfunction of that gene.So, you can look the schematic diagram.So, in forward genetics, we start with the phenotype and then we map the associated geneor the genetic loci which is associated with this phenotype.Whereas in case of reverse genetics, we have gene, we identify the gene first and thenwe try to understand, does this gene has any role to play in the plant development, andif it is so, what is its specific function in the plant growth and development?So, now we will take one by one.So, first we will discuss more about the forward genetics based approaches.So, as I said the first step of the forward genetics is that you should have a mutant;mutant is very important, so or any variant.So, mutant means a particular defect in the development.So, defect is important because if you do not have abnormality, it is difficult to tracewhat is the normality or what is the mechanism which is regulating the normal development.So, typically what what are different steps which we follow during forward genetics basedapproach?The first approach is identifying a desired developmental phenotype or a mutant with thephenotype.Then once we identify this mutant, then obviously, we are going to analyse genetics or inheritancepattern of this mutant whether it is dominant, recessive, semi dominant.And then once we know the pattern of the mutant or the inheritance pattern of the mutant,then we go and we try to map the genetic loci.So, this is important then try to understand that what is the genetic loci which is actuallyresponsible for this developmental defects?And then final step in forward genetics what we take, we then validate the genotype andphenotype association.So, how we do?The first step normally we take a mutant or any variants, natural variants are also availableto study, and then and then we finally, do the complementation to validate this one.So, the first important thing in any study of plant developmental biology is to fix yourbiological question.So, if you recall our previous classes, so what we have discussed, we have referred whatare the different developmental events which takes place during the process of plant growthand development.So, depending on that you can first choose your questions.So, you can define that which aspects of development you want to study.For an example, are you interested in studying what are the genetic loci which is responsiblefor maintaining the meristem, second developmental events what you can study is the organ identityand patterning.So, you want to understand that what are the genes or what are the genetic, genetic elementwhich is responsible to define a particular identity of a organ or a tissue.And the second thing is that you can identify what is the genes or what are the geneticelements which is responsible for patterning, this is important in the process of a plantarchitecture maintenance.Then you can identify mutants which shows abnormality in shape or size of a particularorgan, particular a part of the plant.And then another very interesting thing what you can study what are the developmental mutantswhich affects phase transition.So, if you recall again our previous class, so we have already discussed that during growthand development, a plant passes through different phases starting from the embryogenesis phase.Then in the post embryogenesis first it undergo the process of juvenile development, thenthere is a transition from juvenile to the adult vegetative development.And then from adult vegetative development, there is another transition or major transitionis from vegetative to reproductive development.So, these phases are regulated by in the time.So, what we can do?We can identify a variants or a mutants where this stage or this transition are defective.Another important thing if you recall previous class, we have talked about the activationof branching or activation of secondary growth.So, this both this path processes are regulated and it occurs only at a certain time point.So, it is also important or as you can imagine that they are also regulated by genetic element.So, we can also identify or screen a mutant where these processes are defective.So, now we will go and look what are the different ways to identify the mutant.So, the first step of forward genetics we are taking here to identify a desired developmentalphenotype or desired mutant.The first and maybe more important with respect to the breeding approaches was to look thenatural variation.So, in this nature, there are a lot of variations in the developmental stages, organs, structurefunction everything.So, if you look for example, this upper panel, so here are different accession of Arabidopsisthaliana.And what you look there are variation morphological variations.So, if you look their leaves, the number of leaves, shape of leaves, size of leaves, sothere are huge amount of variation even within the same species.So, what we can do?We can choose any one of these two contrasting two accession with the current contrastingor with the different trait, and then you can try you can try to identify the associatedloci.If you look here, so this is basically variations at the 6th rosettes leaves, so it is acrossdifferent accession.So you can see that even not only the overall morphology, but shape and size of a specificorgan also shows a large amount of variation across these species or across the accessions.Here is the variation in the architecture.So, you can look these all plants are compared at the time of after transition floral transitionhas occurred.And you can see that across these different variants or different accessions, there isa large amount of variation in their overall or final row final plant architecture.So, this could be one source where you can identify a particular trait of interest, andyou can you want to study the associated gene.Second way of moving is you can induce the mutations.So, you can basically create a mutant plants where you can look for a special phenotype.So, there are many way to do that, we are not going to talk more about it, but whatwe can do we can take few example of very widely used way to study the to generate thedevelopmental of mutants.So, there are three major category, chemical mutagens, physical mutagens and biologicalmutagens.In chemical mutagens the most frequently used chemical is EMS; EMS it generates a pointmutation.So, what happens you will see in the next slide that it creates a kind of change thenucleotide in a way that GC base pair getting converted into AT base pair.So, this is a kind of point mutation and this any point mutation has a lot of benefit.Second mutagen is physical mutagens where you can use different electromagnetic radiationsgamma rays, X rays, UV light and they can induce random mutations in the genome.And we will take an example of a gamma rays radiation.Third way of generating mutants are biological mutagens, where we are using a bacteria whichis called Agrobacterium.And Agrobacterium basically we are using this Agrobacterium mediated transformation system,where we can use either T-DNA or transposons based system, and we want to create a kindof gene knock out by insertional inactivation.So, again if you look how EMS works, so as I said; so let us assume here is a GC basepairing.And if you treat with the EMS, what it does; It changes it modify this nucleotides theG and it adds methylene.And once this modification has occurred in the next round of replication this methylatedG cannot pair with the C, instead it can pair with the T. So, now it has added T in placeof C in one strand and once this DNA will undergo the second round of replication atthe place of T, naturally A will be added.So, if we look from here to here there is a transition of CG base pair to TA base pair.This is creating a kind of point mutation.And this is one of the very powerful chemical mutagen which we are using or people who areworking in the field of developmental biology is extensively using to generate point mutations.So, what you do with this one?So, the way of generating let’s take typical example of Arabidopsis thaliana.So, you take a large number of seeds, and expose the seeds with the chemical.And what you are expecting?You are expecting a random mutations in some of the seeds, anywhere in the genome, youcannot control it.And then you generate a population where you will have some non-mutant, some mutant.And whenever this mutation will occur in the germ line and you take this plants grow them.And when you grow them, there is a possibility, if your plant has come from the non mutatedseeds, it will have a normal, it will create a normal pattern, but there is a possibilitythat you screen a plant which is coming where the genome has been already mutated.So, you have generated some kind of point mutations and as I said that this way of generatingmutation is totally random.So, you have to screen a very large population of mutant to find your desire mutant.Then you screen this population and look for your phenotype, fix your phenotype.So, first thing let us assume that if you want to study the root development, then youlook a mutant where root is defective.If you want to study the shoot development, find or screen for mutant, where shoot isdefective, flower is defective, whatever your interest is, you can fix it and then try toidentify a mutant phenotype.And we know from the genetics this mutant can be either dominant, recessive, it canbe either in homozygous condition or heterozygous condition; this all kind of things you cananalyze, and you can understand that what is the pattern.So, I will take some examples.So, if you look here, this is Arabidopsis root, this is a typical or very early stageArabidopsis seedling.When this seedling was EMS mutagenesis and it was and people were looking for some kindof defects in the root, and what we observe that if you look this, the shoot developmentslook quite normal.But there were three independent mutants, which was identified from this populationwhere root development was affected.So, you can see they are very short root.Not only that this is powerful tool.You can even make a very sophisticated genetic screen.For example, if you look these two pictures, so here we want to identify the tissue patterningin a very special tissue which is called phloem.So, if you look this signal, this is a protein green fluorescent protein.And what happens that if you express or if you produce the green fluorescent proteinin the phloem cells, it can move through the phloem cells, and it get distributed in theroot tip.Now, if someone want to identify what are the genetic locus or what are the gene whichis responsible for giving a proper identity to the phloem cell or regulating a properdifferentiation to the phloem cell, you can mutate this plants and look the plant wherethis property of phloem is affected.So, if you look the mutant, here you can see that we have the green fluorescent signalsomewhere here, but somehow the fluorescent is not moving, the GFP is not moving.So, this tells that in this mutant some aspect of either phloem developments are affectedor phloem function which is transporting the molecules are affected.So, you can fix your biological question here, and then you can go and trace back what isthe genetic loci associated with this one ok.Similarly, this mutagenic approach you can take, let us assume that you already havea mutant, you know function of a particular gene through the forward genetics.You can take further and you can try to identify other genetic component of that entire pathwayby inducing by mutagenizing your mutants and screening for second site mutations identifyingenhancer, identifying suppressor.So, you can do.take this examples here.So, this is a gene which is called AG AGAMOUS gene.So, in this mutant, this is a single mutant, you can see some phenotype, a phenotype isindeterminate floral meristem no carpel, but stamen is fine.When this mutant was mutagenize and looked for another second site mutation, so therewas another mutant which was identified hua 1, hua 2; and these mutants along with theagamous 4 mutants, double mutants.If you look all other the mutant phenotype is still there, but even stamens are gettingconverted into petal.So, either second site mutation can enhance the first mutation.Similar case, you can here hua 1, hua 2, alone they do not have any phenotypes.So, if you take a single mutant, there is no obvious phenotype, but when you screenfor another higher order mutants, so hua 1 and 2 with hen 1, there is a loss of floraldeterminacy there is homeotic conversion of stamen into petal.Similarly, hua 1, and hua 2 with ago 10 gene there is loss of floral determinacy, withagamous, there is loss of floral determinacy and conversion.So so, if you look this this is a way to this provides a very powerful way to perform avery special or very targeted genetic screen and identify double mutant, triple mutant,and then try to understand the entire genetic pathway you can trace out the genetic pathwaywhat all genes are responsible to regulate a particular process of development.Another important thing if you look here this is a screening a suppressor.So, let us assume that I have identified a mutant where leaf is defective.If you look here this is wild type there are so many leaves and shape and is different.But when you have a mutant where number of leaves and the shape and size of leaves arealtered.Now, I have identified one gene or one genetic component which is responsible for this patterning,I want to identify what are the other gene or what are the other genetic components whichare interacting with this gene to provide this particular shape and size.You can take this mutant and now you can look for double mutant where even the previousmutants are getting suppressed.This is called suppressor mutants.In these cases, you are adding them and you are identifying more severe mutant phenotype.Here we are looking for a mutant where even the existing phenotype is disappearing orgetting supressed.So, this technique you can use to identify both kind of genes.Ok I will not go in detail, but you can look this picture here gamma mutagenesis, physicalradiations being used to mutagenize the plants.And what you can see that there are lot different kinds of mutants with different phenotype,you can see chlorophyll phenotype, leaf phenotype, shoot phenotype, flower phenotype, all thiskind of phenotype or mutants can be generated with this mutagenesis.So, these are the two way of doing mutagenesis.And third another very very important way to generate a desired developmental mutantsis biological mutagens.Here there are three major approaches which is being taken place.First one is T-DNA mutagenesis which is done by Agrobacterium mediated, T-DNA insertion.Second is transposon based mutagenesis and third is activation tagging based mutagenesis.So, in transposons we all you have studied in your course or previous course that transposonsare the mobile genetic elements which naturally present in the in the organism, and they canactually jump in the genome that is why they are all also called jumping genes.So, we can use them as a technique to basically disrupt a particular gene and identify themutant phenotype.So, what we can expect through this one?So, here basically this process is called insertional inactivation or insertional mutagenesis.So, what happens?So, either you take transposon or you take activation tagging; activation tagging isslightly different, but if you take T-DNA or transposon.So, what basically you are doing, you are allowing this molecule to randomly get insertedin the genome.And if it happens that, it got inserted in the gene or the regulatory elements of thegene, it can affect the expression or function of the gene and that results in some mutantphenotype.So, these are some possibility.Let us assume that if this got inserted in the coding region, it may disrupt the codingregion and you might get a loss of function phenotype.So, where the gene is disrupted, no functional proteins are made and that is why you willhave a phenotype because of the loss of the genes.Second possibility is that if the insertion somehow happens in the promoter region oruntranslated region.So, promoter regions you know this is very important to regulate expression and expressionpattern of a gene.UTR regions - Untranslated Region, they directly are not involved in coding the protein, butlot of regulations occurs through the untranslated region.So, if there is insertion or disruption because of the T-DNA or transposons in this regions,what we will expect?We can expect either reduced expression or altered expression pattern.And in both the case, we might have the phenotype.Another way is that it happens that somehow the insertion happens in a gene or in thegenetic elements and it can also enhance the expression.This is more relevant when we talk about the activation tagging, but it has been also seenthat if insertion happen somewhere in the promoter or it happens in the element whichis like silencer or repressor of a gene.And if this insertion suppress or kills the regulators, then you might have increasedexpression.But again as I said that this processes are totally random.So, there is also possibility that there is a multiple insertion in the same genome atthe multiple site and this may generate a multiple knockout or knock down whatever isthe condition ok.So, we will briefly discuss how T-DNA mutagenesis occur.So, Agrobacterium is a bacteria which basically is known to infect the plant cells.And it has a plasmid which is called Ti plasmid, plasmid you know it is extra chromosomal geneticmaterial.And what happens that when the bacteria infect a plant cell, a section a portion of thisplasmid Ti plasmid which is called T-DNA is transferred in the plant cell.And then this T-DNA get integrated in the plant genome, but that integration is totallyrandom.So, it can go and integrate anywhere.So, if you want to use this technology, and if you want to create and there are certainelements which is may be more relevant in the making transgenic, you might discuss infuture.And what happens here that if you can genetically engineer this T-DNA, you can basically usethis technology to generate mutants.So, what you do, in this left border and right border you can put either a visible marker,this will help you to identify the plants where insertion has occurred or you can puta kind of selection marker.Selection marker, you can put a antibiotic resistance gene.So, you can basically directly select the transformant using this antibiotic.And then use this and generate a large number of a transgenic plants.And then screen them either based on the visual selection marker or by selecting on the antibiotic.And this has been very extensively used in model plant Arabidopsis thaliana.And in fact, of full fledged collections of this T-DNA insertion plants or mutants hasbeen generate, and it has been stored or maintained as a firm of a big program where anyone acrossthe globe can order these lines, they can use for then their own specific study.So, if you look how they look, so typically this is how it happens.So, if you take one example this is one gene, and what it has been found that through theprocess of T-DNA insertion, multiple site insertion has been generated.So, in some mutants, it was inserted here; in some mutants it was inserted here; here;here; here.So, you can say that for this gene at least 6 allele has been generated through the T-DNAinsertion and all the 6 mutants has phenotype.So, if you look this is a normal wild type plant, and the mutants this is 1, 2, 3, 4,5, 6, depending on their site of insertion, there is a variations in the phenotype.So, all these mutants they can be simply generated by integrating the T-DNA.So, second way of generating mutation is transposon based mutagenesis.One very commonly used transposon is Ac Ds based.So, what happens Ds is the element of the transposon which actually jumps which getexcised from one place and get integrated to the another place.And Ac is the part which helps in the process.So, what you can do you can generate a Ti plasmid you can engineer Ti plasmid and youcan put Ac here in one Ti plasmid in Agrobacterium; you can put Ds in another Ti plasmid in theAgrobacterium.And what you do you can generate independent transgenic lines one for Ac elements one forDs element.The important thing is that Ds element cannot jump until and unless Ac element is present,but the good thing is that Ac element can work in trans, so they need not to be together.Even though if they are present in the same plant at different genetic loci, it is fine,they can still work.And then what you do you take this plant; this plant, and if you cross them you arebasically bringing about Ac element as well as Ds element in the same transgenic plant,and once this Ac is available this Ds will start transposition.And during that this transposition it can go and it can inactivate or it get insertedor integrated at different genetic loci and which might create a developmental mutant.See the example here.So, using this example, this is maize plants, some mutants had been generated.If you look the wild type plant, these mutants, the leaves are not looking normal.This is more kind of drooping leaf they are falling off.Ok, another very important transposon which was used which is being used for generatingmutation is Tos 17.Tos 17 is a retrotransposon and it is very important because this transposon normallyis stable at the adult plant or at the later stage of development.So, once it is the element is present, but it is not very actively undergoing the processof transposition.But if you generate a callus from these plants, at callus stage this transposons get activatedand it is started jumping.So, particularly for those plants where we can generate a transgenic plant using callus,it is very good way to generate a lot of insertional mutation of for this one.Rice is one very good example of it.So, what you can do, you can make a callus; in the callus the transposition will occurdifferent genes or different loci will be disrupted.And then use this callus to just simply regenerate through the process of tissue culture andmake transgenic plant.In these transgenic plant, you can identify a developmental defect, developmental mutantand you can generate collection of such kind of mutants.Here is few examples in rice which using this Tos 17 retrotransposon lot of mutants hadbeen generated.This I am showing only few of them.You can look here this is thick culm.Then you have a kind of abnormal flower, you have endless tiller mutants, you have a verydense panicle, panicle is basically inflorescence.So, dense panicle means you have more panicle branches, more number of flowers, more numberof seeds.So, this all mutants they have been generated by using Tos 17 based retro transposition.Both have these approaches normally the Ac, Ds based approaches or T-DNA based approachesnormally or usually they generates loss of function mutants, because they might killthe genes they are more prone to inactivate a gene.But another way to a study a gene function that if you can enhance the expression ofa particular gene, and how can you enhance?So, here this is called gain of function.One way of enhancing is that you do not change their domain of expression.So, they still express in the same tissue, where they normally express, but their expressionlevel goes up.Second way is that if you change their domain of expression or you can make them to expressin a cells or in the tissue, where they were not expressed earlier.And if this kind of genetic regulators are sufficient to give a proper identity or properdevelopmental events, if they are sufficient to initiate that kind of program, they willgive a different phenotype.And this is being used through a process which is called activation tagging.So, what happens in the activation tagging, random insertion of multiple enhancer.So, enhancers are the genetic elements, which can increase the expression of a gene.So, what people are doing?They are using multiple copy of enhancer and they are genetically engineering T-DNA ora Ti plasmid and they are using this enhancer through Agrobacterium mediated transformationand they are generating a large number of plants.And then there are those plants they are looking ok, if there is a plant where there is a phenotype,and most likely this phenotype could be because of the activation of a gene.So, if this enhancer elements, let us assume it gets inserted somewhere here very closeto promoter of a particular gene or the regulatory elements of this particular gene, there isa possibility that because of its enhancer activity, it will enhance the expression ofthis gene or this gene.And you might have a developmental phenotype in this case.This is one example where activation tagging has been used to generate mutations.As you can see this is the site of mutations and this mutations can actually increase theexpression.So, if you look this is a wild type, this is RT-PCR, we will discuss some time in anotherclass, how we do exactly.But this is to measure to quantify the messenger RNA level of a particular gene.So, if you look this gene and this gene, and if you take the expression label in the wildtype, these genes are expressed at a low level, but when there is a activation tagging orinsertion of the enhancer element in between you can see the expression level of thesegenes are significantly increased in these lines ok.So, we will stop here this class, in the next class, we will take this further and we willdiscuss if we have a mutant how can we go further and try to identify the genetic lociassociated with it.Thank you very much.