Metabolic Engineering of Anthocyanin Pathways for Novel Flower Colors

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course on pharmacognosy and metabolic engineering this is lecture 55 uh so where I will discuss metabolic

engineering of Anin Pathways in flowers so in lecture 54 I have discussed in details about the anthos in biosynthesis

the chemistry structural diversity how simple chemical transformation hydroxylation and methylation can

convert one Antoine into another their stability and how they are transported into the vac and as a very exciting

metabolic engineering application with expression of transcription factors in leaf leading to produce huge amount of

anthocyanins in tobacco cell cultures so now here we will talk about the metabolic engineering attempts made with

the flower so this has tremendous industrial application because uh ornamental plants there is a very huge

market and uh as you see that there are multiple colored flowers are coming but even with the traditional breeding

everything cannot be achievable particularly the blue coloration is a challenge like if you can make blue rose

blue cordination so there is a huge market so unfortunately the achievements was not that much but still there are a

lot of hopes there so scientist has cracked the major bottle neck and I think years to come we'll be able to see

blue flowers blue rose flowers so now go to the next slide so the concepts to be covered bios synthesis of anthis I have

already discussed manipulation of anthos pathway that is our concept that is our theme of discussion in this class so I

will talk mostly about that yolow flower by redirecting flavonoid biosynthesis so here uh and then making

blue flowers and then attempts to make rose with blue hues so again this scheme now I have

mentioned this scheme several times so I can avoid so antoin C6 C3 C6 structures so and this scheme also I have discussed

in the previous class in pretty details so only thing to mention here that these

are the target genes uh which need to be either uppr regulated or down regulated leading to

the formation of enhanced or suppressed antoin accumulation so C CHR is a new one so

CHR will come in due course I'll talk about CHR so if you remember CHR I have mentioned particularly in the context of

isoflavonoid pathway where CHS and CHR work together and which which produces ultimately the

isoflavonoids okay now modification of anthos in amount so one uh uh Target was set to make basically the

brick red or orange red uh pelargonium so transgenic oh no sorry

ponia so ponia colors brick red or orange red ponia colors are rare So scientist what they have attempted

basically they have attempted to uh hire a maze dihydro uh dfr dihydro flavonol

reductors so a mage dfr to be transferred into the ponia and to see whether it makes any effect on uh

coloration so uh like uh that is one uh or also the F3 Prime H F3 Prime 5 Prime H can also be targeted so what we see as

it is expression of CHS and chalone synthes in ponia many purple plants resulted in few plants with pale color

or pure white color so this is anti- sense so when means that when you block the chalone synthes that is the first

step so that means you are blocking the entire pathway so coloration will be theoretically suppressed and in in in

practice also it happens and uh but uh Sometimes some transcription Factor genes like me R genin when they are

transferred in tobacco that making more intensely colored p ments because that transcription Factor has some role in

upregulating the Anin pathway similarly antisense suppression of flavonol synthes gen in ponia also resulted in

higher anthos in content so that aspect maybe will come later so here is basically the modification of flower

color in ponia by expressing dfr and dfr is from the mze and as a result of that what we see

that the color of this turn different uh okay so transgenic cultiv Express the mze dfr Gen which is showing

here and B1 is the red fling ponia cultiv that accumulates sidin type of antoin so uh uh so this is basically the

dfr and dfr basically part it converts I hydrol to this uh eventually to the the ponin glucoside and if it accumulates it

makes this color so okay and uh so uh this is and this is reported long

back in Trends in biotechnology and this is another one where

the the bottom four are as a result of transgenic application whereas a to d the a b c d these are basically

non-transgenic maybe it's it's created out of the mutation but whereas this is all these are also ponia so modification

of ponia color by genetic engineering so this was also reported long back in the famous jard PL cell so here basically in

case of e if you see now we can see one by one this

one so here what happens the uh antisense CHS when I I mentioned this just now that antisense CHS blocks the

entire pathway almost so no coloration leads to White now second one is basically the

transformed Rose dfr Gene and as Rose dfr Gene basically Rose is basically sinine so the dfr converts what the D

dihydro quatin to lianin and subsequently to cadin and as a result of that is it is developing the red

coloration okay and the third one is basically they have transfer a gene which suppress both

F3 Prime 5 Prime H and and if and flavonol synthes activity so as a result of that what happened

that dihydro cerol will mostly accumulate and that dihydro temperal may

leads to the formation of honin and as a result of that you will get this orange red color okay and the fourth one is

basically transformed with a sense ponia F3 Prim 5 Prime H so when ponia F3 Prime 5 Prime H is transferred then what will

happen that uh that leads the pathway flow more towards the delphinine but uh if the

background is with if if the if the plant transgenic plant the the explant used is already containing another Gene

of say uh which led to the formation of redish color then with the blue color what will happen these two will mix up

and as a result of that there will be different coloration so if you see this these three these have three different

coloration the strategies are different one antisense one OV expression one another Gene so as a result of that if

you if want play with this pathway jeans it is possible to make the changes in the flower coloration so uh this one I

skip it now come to very important and interesting thing like normally what is happening malony qu joins with three

molecules of uh three molecules of malony qu joins

with one molecule of paracom qu and that converted into tetrahydroxy chalone and the enzyme is chalon synthes

CHS however as I said in case of this isoflavonoid pathway so there uh CHS

work with Chi but there Chi is not there but instead ch R is there so in other word uh in this

case a pathway will move in this direction where

where CHR and CH s will work and leading to the

formation of uh six Prime desoxy alone this is is not very clear here but this is six Prime desoxy chalone now what

happens that six Prime desoxy chalone is not the natural chalone that accumulates in the flower okay but it is possible

how if you take a medicago Satia which is basically a legume I mentioned try to find the relationship

what I said the legume produces isoflavonoids and isop flavons and there the CHS work with CHR

so that is why scientists they have hired the CHR from medicago SATA okay so and that they express in

ponia so what happens in this case the uh so the pathway now will moves in a New Direction and eventually uh the the

CHS work CHS will make this uh tetr hydroxy chalone that tetr hydroxy chalone cannot really converted into

naringenin instead what I said the pathway will move in this direction okay and then what will happen

that this leads to accumulation of six Prime desoxy chalone and this is possible if you express the genes under

the control of a strong promoter there will be more CHS as compared to Chi so therefore the whatever this tetr hydroxy

chalon will be produced that will be channelized towards uh six desoxy chalone because of the new

enzyme which you have introduced and uh and the beauty is this this is a new compound and it is unnatural so the

petunia cells cannot metabolize it further so it started accumulating once it started accumulating what it will

what you will see you will see the accumulation of yellow coloration so accumulation of yellow

coloration okay so this is what is called redirection of flavonoid bi biosynthesis so flavonoid biosynthesis

normally moves in this direction now you are blocking that and allowing this to move

in this direction so that is how it is called redirection now let us see the result as a result of that so they have

used two type of ponia one is colored one is white so white is Asic Asic not Asic means colored so Asic is uh the one

you see this is C left is the control this is control okay and uh and then they have

expressed the genes and as a result of you see the changes in the coloration so this is

control so if you and this is the test transgenic this is the transgenic so what you see that you compare to this

and this the yellow coloration is noticed here here yellow coloration is quite intense and here actually three

developmental stage the left uh one is control uh so this is this are control and these are the

transgenic so the product uh so the yellow coloration it is possible to produce in a Asic background that is the

white ponia if you take uh and then the if the gene is transferred and expressed properly then uh this white ponia will

will will produce upon gen expression will produce the yellow coloration but if you take

the cyanic background then what will happen that already it has a color now uh when you have done the paint brush

work means with with with paint and brush in a paper so when you a color is red is there so you add a yellow what

will happen the original color will become diluted so similarly that is what is noticed here and so the color the

original intense color will get diluted as a result of that you'll get different pattern of coloration here and only

thing is that I will uh mention here the uh when it is redirection that means more

of chalone is produced isn't it so what you see here the chalone percentage in this line is

51.7 chalone percentage is this line is 62.5 okay uh so that means more chalone is basically producing more more chalone

is accumulating when more chalone is accumulating so it's producing yellow dis oxy shalone so that is basically

mixing up with the other antoin and as a result of that if the pH is favorable as a result of that it makes different

pattern of coloration and some of these colorations are indeed novel which cannot be achieved by traditional

breeding so other example is this metabolic engineering to modify flower color one is

the uh ponia they have used another variety purple variety and they use antien

separtion of chalone synthes and as a result what you see depend this is the control which produces this color when

you supress depends on the level of expression you see that coloration changes and eventually

it produces white flower in different lines depends of the expression levels how many copies of

genes inserted all these will influence how strength is your promoter and the second one is this that here basically

they have uh transer ponia are rose dfr and as a result of that what will happen uh the novel flower color in the

left side this is the transgenic and this is the uh no this is transgenic sorry this is

the control so the novel coloration was developed as a result of that the rose

when the rose dfr was expressed in ponia okay so this is Noel and third one is this that here

the and uh this is the control it normally produces this bluish color this is

Toria which produces bluish color and uh this is a sumar wave a particular variety and what they have done

basically they basically made the sense suppression of CHS or dfr as a result of that you see that which is more more

bluish purplish color now it it changes it coloration so some portion is white some portion

is blueish so that means a novel coloration pattern develop as a result of sense separation of CHS and dfr genes

so and this was report Ed uh in some of these were unpublished those days when this review was published from there I

have taken so this is fine but ultimately what I said at the beginning of the course is that beginning of this

lecture is that eventually we aim to make blue flation blue color so if you can make novel blue color particularly

in this cation or in this torania or in Rose you will have a very good Market Horticultural market so but in order to

produce blue flour that it has to meet several conditions one is the synthesis of three prime 5 Prime hydroxate antoin

that is required that means delphinine so three prime 5 Prime H activity must be there and also there is a need for

present of flavonol copigments so flavonol copigments are required for example that delphinidin

also you also require the myin to be present there so when myin to be present so that is basically the flavonol

copigments so that will work and in with the delphinidin and makes the stability of the coloration and another important

point is that I have mentioned the high vacular pH if you remember previous one of the previous slides I have shown that

blue coloration means between four and five so that is important now flower petals are they are actually meant uh to

have lower pH in order to make the stable anthos so making a higher pH is also a challenge and and then also if 3

Prime 5 Prime H activity asile transfer activity as also necessary for producing the blue color

now what what has been achieved is this that this is the requirements

and this is the achievements and the bottlenecks achievements this the genes encoding F3 Prime 5 Prime H has been

isolated from petunia eggplant and many others and a transgenic Violet cination was successfully developed by florigan

limited and Sant limited both are this one is Australian another one is Japan based companies so where they have

introduced a ponia F3 Prim 5 Prime H and dfr gen into a dfr def deficient white cination so white cination is where is

the dfr deficient that means dfr genes are not working there so that is why the if dfr is not working then you know what

is happen dihydro kemperol will accumulate so D only D if dihydro kerol accumulates it if it cannot move towards

ponine or towards uh delphinine or towards sin in what will happen the color of the flower will be white

because it stops there so they have taken a dfr deficient white cination and there they have introduced F3 Prime P

Prime H and and then as a result of that they perhaps they have also hadir uh another dfr from other source and as a

result of that they see changes in the coloration now now apart from this these are the some achievements but still some

bottlenecks are still there that is the control of the vacular pH so vacular pH only few genes have been cloned so which

really uh maintains the proper vacular pH so that you get the stability of the coloration and uh so this is what is

that the mou coloration develop by introducing a hf1 and dfr gen in white flower cination

variety Mund dust and which I said that why and and and that accumulated dihydro cerol so when

dfr was used so there here dihydro cerol accumulates and what happens that this one normally dihydro if accumulates it

makes uh white color but here particularly when they have hired a dfr from different sources this basically

leads to the formation of a novel color in this uh coordination okay and next is this this

is the uh transgenic Violet Violet cination produced delphinidin type of pigments and this is because they have

transferred F3 Prime 5 Prime H and the there are two the left one is the P one so this one is the paler one which is

called Moon dust and this one is the darker one which is the Moon Shadow and this is the moon dust

and with further advancement it was possible to make some blue hues in this cardition as well as in Rose which was

not possible by traditional breeding so next attempts were made by this hun and uh Floren group they work together and

they really aim to make uh blue colored Rose so blue colored Rose as you see from the knowledge what

we have gained that in order to have blue color rose the delphinidin must be accumulated so

if delphinidin to be accumulated then f3.5 should be work and along with that the

mtin myin is flavonol pigment so myosin accumulation is also required okay and uh apart from other so native Rose only

accumulate ponin and sinin based antoin that is uh so what is

this so either ponin or sinin based anthos sinin okay and delphinidin based Anin

basically there is a deficiency normal rows of F3 Prime 5 Prime H that is if it is there at all it is nonfunctional so

it is important that the F3 Prime 5 Prime H to be hired from a heterologous source so that and if it is expressed so

that dihydro cotin is a common one so the normally dihydro cotin for cine it moves

in this direction or this but then with this the pathway should be able to move in this

direction and as a result of that if the rose petal generates delphinidin and again there are lot of

factors only delphinidin will not help along with that mtin should be produced in higher amount along with that the

stability pH uh checked then only so therefore Gene is available so therefore in order to

have a Target Rose cultiv where the gene to be transferred it is important to do a thorough screening to find out a rose

cultiv uh where uh a rose cultiv accumulates more flavonol as Co pigments like my

resetting so with screening it is possible with screening of the flowers of hundreds of cultivar it is possible

to pick up couple of 3 four five uh Rose cultivar where which are reach in Martin flavonol so that is also another Target

and next Target is that to check the vacular pH petal pH to check where uh the pH is in highest sight so with the

thoros screening the the scientist of from these industries are able to find out at least 10 uh Rose cultiv which

actually speaks in favor of their work that means uh where myin content is more along with that the vular pH was in the

higher side so that was important and now what they have done so now they have made different constructs so one

construct is overe expression construct where F3 Prime 5 Prime H they have hired from Viola Viola the name itself

it's that it produces violet color so when there a violet color then F3 Prime 5 Prime H is there so that they have

hired there and they have hired another asile transfer uh from Tania so this construct they called PSB

130 and uh when this construct was expressed in rows what they found they found changes in coloration so the left

is host right is transformant host means you can we can write control host okay host and this is

transformance so you see changes in the coloration so when F uh 3 Prime 5 Prime H is expressed the

deep red color the deep red color is now getting dilute towards more pinkish here also it is

getting in this color okay here is slightly different we to explain here it's more towards

bluish okay uh and here also it is quite clear but not everything cannot be explained so this is what they have

published this they have published everything in this particular uh paper so we'll come to that so this is one so

they got some success with this so next what they have done they also attempted to make

uh only with a viola and see but that result was not that interesting next what they have done they have used Iris

dfr and Viola F3 Prim Pi Prime h two gen together so that gives a better uh one than the previous but uh what happens

next what they thought that the main culprit was the rose dfr so what happens even if you put the dfr

and so what I said here that what you are hiring

uh you hiring a dfr you're hiring a dfr n 3 Prime 5 Prime H but what happens uh okay you

adding a dfr this one sorry sorry uh I will erase this one not what will happen that

this particular this particular dfr is already there in the row so when you are

hiring F3 Prim 5 Prime H and dfr from two different plant putting there but this dfr which basically convert dihydro

cotin which is the major intermediate from there only it converts di hydromine if this dfr is strong the red one which

I am showing here if this is have a more stronger expression so what will happen it will try to pull the substrate more

towards sinin even if you put extra copies of F3 Prim 5 Prime H and dfr from different sources with expression the

endogenous dfr will not allow it to function so the strategy what they have learned from this this this that the

third strategy what they have done is that they have used a RNA I approach to block the rose dfr so the rose dfr to be

blocked using RNA that is why I have shown in this way this is basically a hairpin construct and I think hapin

construct I have mentioned when I talked about the uh this uh this Morphin alkaloids so in

that or that that time because now because of shortage of time again I will not discuss that uh so you can always

revise from there so now so the the number one let us see first one is that they had hired Idis dfr that is good

they have hired uh Viola F3 Prime is good and the third strategy is basically they they

they block the rose deer Now situation will be favorable so that means the if I say

favorable then this will not work anymore uh so if this will not work anymore cyanidin formation will be will

be significantly reduced instead the hydr settin will be move towards the hydromine because you have hir

a viola what you have yeah you have Viola F3 Prime H and you have also hired a Idis dfr and as a result of that the

the pathway will move in this direction and eventually it produces delphinine and this as I said this is

already producing in higher amounts so this plays an important role in uh stabilizing the

coloration okay so this is the best construct with this best constructs what they have achieved so

the they have achieved a coloration like this and where the 98% was

delphinidin so the flower color is evidently Bluer Than That of the host and the commercial varieties of this

Violet uh and this is regarded as the bluest of current Rose varieties so you see this

color and some of the transgenic rowes derived from this also exhibited P color containing decreased amount of anthos

so so what happened this was the target uh and that now produces this so this was the cultiv

which is producing this sort of coloration one thing you see this coloration is not the this is

slightly P red this is slightly P red they have purposefully chosen such one so that uh the red will not interfere

and that they have blocked the dfr rose dfr they have add the Irish dfr and add the viola F3 Prim 5 Prime H so that

leads to formation of this coloration and they claimed in this paper that this was The Bluest so far achieved and this

is really an outstanding work and this was published in plant and phys cell physiology so after that uh I have not

come across with further work maybe because it's a industrial work so there is a confidentiality to be maintained

and patent issues and other thing once those were sorted out and definitely they will publish so maybe we have to

wait for few more years when we'll may see a deep blue coloration row available in the market with this I end this class

and this is all about metabolic engineering of uh anthos pathway and you see lot of positive results with this

and the Beautiful science behind and this that also I have explained with this I end this class thank you