Biosynthesis and Transport of Monoterpenoid Indole Alkaloids in Catharanthus

[Music] [Music] welcome to nptl online certification

course on pharmacognosy and metabolic engineering this is lecture number 30 where I will discuss about the model

for biosynthesis and secretion of monot Indo alkaloids involving multicellular uh multiple subcellular

compartments so uh in the last class I have briefly discussed about the cellular

localization of both the enzymes and intermediates in

uh uh which are involved in the monot Indo Al production such as vindoline and catharanthine as well as serpentin aalin

and so on and these two joins together by the action of parides and forms the VIN blastin and also I have shown a

cross-section of the leaf uh where different uh cells where I have shown the

uh localization of different cells uh such as laifer cell such as uh idioblast cells so where these uh indol alkaloids

accumulate or in other word the late stepes of Indo alkaloids are operating there whereas the most of the stepes of

Indo alkaloids are accumulating in the epidermal region and also I have mentioned that this laifi and idbl BL

these are the cells which accumulate latex and the uh last monotoring IND alkalides

which are apparently more toxic so that basically stored in the latex so that it will not hamper the normal cells of

catanas Rosia cells and lip cells so with this what I'm going to cover so I'm going to see uh cover again the

compartmentation of tarpo IND alkaloids in C Rosas so I'll draw a line diagram and here particularly I will

emphasize the role of epidermal cell in uh in in modulating the pathway and once I finish that then I'll will go to the

cell specific localization of tarpo indol alol that is linked with this and how this indol alkaloids are basically

transported from one typ type of cell to another and then finally I will speak uh on uh a specific transporter

which was characterized few years back so that plays an important role in transporting one of the central monotor

indol alkaloid intermediates to vacu so with this now let us go to the board and I will draw the diagram so

this is basically uh biosynthesis of monotor indol

alkaloids involves multiple sub

cellular compartment and cell types so this is more or less the

continuation of the previous class that is class number 29 Okay so let us draw uh quickly I draw

a schematic plant diagram of a plant cell so here this one is a mesop cell which I

put it in green color and the other one which I put now in light

green color that will denote the Lea epidermal cell so basically this is

leaf epidermal cell and this one is

the ESOP cell and the other region what is

outside that is basically the cell surface so and inside this

mesop I am also going to show this two specialized structure one is this idioblast or

laifer and also you have the uh one one specific cell exist which is

called FL Pena uh and then

inside in one of the cells it contain the Plus this

is chloroplast okay now very

briefly I project this so for

example the map pathway is operating inside the plastid and that ultimately leads to the formation

of janal which comes out and

eventually it makes 10 hydroxy anal and

then this will move out from this mesop and enters into the leap epidermal cell

where this will be converted into loganic acid so this is a multiple step

process and then it makes seanin and that joins

with triamine which comes from tryptophan

and that makes strictosidine and this strict

toyin subsequently converted through common

intermediates into different products so one particular root makes

tonin and this tonin finally Mak 16

myoxy tonin and that moves inside

the ESOP C again and there this is again

chloroplast and 16 methoxy

tonin enters into the chloroplast and by the action of

nmt it forms this finally the de atile [Music] vindoline

and from here it moves to theblast and laifer

cells uh so here there are involvement of multiple enzymes okay for example d a and d4h is first and then uh D and

that leads to the formation of vindoline

so the enzyme nmt is here this is Str this is

TDC this is uh

SLS and so on okay then uh

T6 H2 then t16 omt and so on

similarly from strictosidine so another Branch contributes towards the formation

of catharanthine so this cenin I'll put it in a different color c

a t h a r a n t h i any so these cenin

mostly this is the most important part mostly it moves out the cell and retained in the cell

surface and only a portion of

cenin can be transported from here through here to here

so and that then that eventually joins and make the diic Indo alkaloids so interesting part is this this this

catharanthine is getting transported out from the cell into the cell surface and here I will talk about one specific

transporter that plays an important role in transporting this out which is these are the transporter molecules

here so these are CR TP

T2 this is the transporter so cathan accumulates in the leaf surface and along with this uh in

the leaf surface you will find lot of works very long chain fatty acid plus

this uh aqt works these are accumulates there

now question is that what role catharanthine plays and second point is that although sufficient catharin are

produced in the cell but most of the catharin are transported in the self Surface and only limited cenin are

available for carrying out the reaction leading to the formation of Vin blastin by joining with the vindoline and that

is one of the reason that why the yield of vinblastin is so less so the next question is that why catharanthine

accumulates in high amount because catharanthine basically protects the leaf from

fungal attack fungal infection along with that there are certain minor insect attack which can also be prevented by

the catharanthine so catharanthine is basically a very important defense compound okay so uh the one way to

isolate cathan is basically one can collect the leaf and then the leaf to be immersed in chloroform for around uh 10

minutes and then what will happen all the catharin will be uh transferred from the leaf surface into the chloroform and

later later this can be purified and collected so that is one of the way which cathan can be collected and that

catharin can be used for doing the joining right okay so and uh another important Point what is import uh what I

must say here that sometime wounding happens in the cell

and then when wounding happens that basically stimulate the uh V Vin blasting

formation uh so wounding stimulates

B indol alkaloids formation this Sy means this

D so this is all about the involvement of lip epidermal cell and the mesophile cell so the mesophile tissue rather I

will not write the mesophile cell I will write here the mesophile tissue that is a better word because it consist of uh

three different cells one is this idas latier one is the normal mesop paliside parena and and another

one is basically the flu parena so this is the

Alid Vena okay once this is clear and I have also mentioned that the idioblast

contains the enzymes like d4h and d and leap epidermis contains most of the enzymes of this

pathway okay so now with this I move into the next point of our discussion let us go to the slide so cell specific

location of tarpo and IDs in catharanthus lift tissue so here I will now try

to uh put my points so this is all

cell specific localization of tarpo Indo aloid in karanas Rosas lift

tissue so here one important point to mention that uh the

vindoline particularly uh which accumulates in the aial part that is the uh up ground Parts

which contain the green tissue right so and you will always find the VIN Christin Vin blastin only in the green

tissue now the content of the alkaloids what you will find in the stem and what you will find in the leaf it

differs and what scientist they have found that the leaf tissue contains more alkaloid and as the leaf tissue matures

the level of accumulation is more so from there scientists they try to work out

that whether there is any relationship between this Leaf development and maturation and the alkco

synthesis so uh this will be clear from a bit of drawing so like this is the

area of Li primordium and here

the red indicates the vascular tissue and uh from here the

leaf originates scientist how they have localized this alkaloids that is very

interesting so they have used the uh mass spectrometry that is a technique

called Imaging Mass

spectr matry along with single cell Mass

spectrometry so using these techniques scientists were able to locate the even the density of the alkaloids that

accumulated in the leaf tissue for example say the alkaloids which accumulated in this

area it's shown in this Turkish blue okay and as the leap expanded the level of alcoh accumulation

enhances so this has been demonstrated using uh these techniques and this is basically showing the

expansion of leaves so there are two particular type

type of cell one is the LI primordium cell and one is the elongating cell so

uh if I make a drawing so so the bottom one which I what I am

drawing this is basically the ID blast cell and the top one what I am drawing so this is basically

the epidermal cell so B I use the same color

here so this denotes the and this denotes

the elongating cell so I will not draw the pathway in the primordium only what I would like to

tell here that here the level of vindoline is very

low whereas here the level of Bolin is high along with

that VIN dorosin is also produced which which is

basically deoxy vindoline and what I have shown in the previous class that uh this the top one is okay one more thing

I have to mention here that this is the EP daral

cell and the bottom one is the

video blast or ltis c

for cell so here uh

the from taronin for example so that that means this acetoxy windol

in and the enzymes responsible for these reactions are this D16 H2 then uh D16 om T

then P3 t3r then n m t and so on and then finally desoxy

vindoline it enters from here into the uh into the later or blast cell and then that will be

this and aoxy Vin now my point

of emphasizing this to you is this that how this enters here so how this enters here the question is that whether there

is any existence of a transporter that

means to get these things okay I use that means to get these things out from

the uh epidermal cell and to enter inside the uh ltis refer cell this require

specific Transporters which are not yet characterized

and so this is very important and then subsequently this by the action of which I have said

many times that d4h and d8 it makes similarly through another pathway from

tonin this induration is produced so the point here is this how these uh uh metabolites

are transporting out from the epidermal cell and entering inside the idioblast cell so that is not known and that needs

to be uh discovered so that that plays an important role further for this

metabolic engineering application so and this one is uh and the LI primordium

it has the similar drawing so I'm not drawn but what important is this that vindoline content is much more here

whereas vindoline content is less here so that is also indicated from this drawing and this one uh this paper was

discussed uh by y

Yama Moto at all in this journal publication new phyto

ELO g i s 2019

uh volume 224 to 848 to 859 so here in this paper they have shown very interesting Mass

spectrometry images so uh when I will make an overview of total indal alkaloid so I will use the slide and there I will

show you those images so let us go to the last part of this class so this is also in connection with with this one

which is the an NF npf transporter so the last part is basically I am I'll very briefly talk about a

transporter this transporter is called n PF transporter this transporter was

shown to exports uh a

central monotoring indol alkaloid intermediate

from the vacle so and this transporter has been

characterized so let me quickly draw a diagram to explain this so this is basically the diagram of the

cell so here I will bring the issue of this uh early steps of IND alcohol that is

seol loanin and

tryptamine and that makes strictosidine so this is the

cell and here with this color I mentioned that this is basically the

vacle so this is vacle v c o l and the remaining portion is the cytosol or

cytoplasm okay so cyanin and tryptamine

they enters into the vacle and this must be mediated by some P

transporter and these two once enter into the vacle these two joins together and form

the molecule strictosidine okay this troiden once it is produced so these

stoin will be transported out from the vual into the

sorry into the cytool where this strictosidine will be undergoing uh strict toid

in uh undergoing uh Detachment of the Su glucose leading to the formation of of

strictosidine a glycon and which will be

subsequently utiliz for running multiple Pathways leading to Tarpin indol

alals so these all we know we have said that this is SGD this is St

Str but what is to be discussed now is this one that is a transporter here which transports this out and this is

the CR npf

2.9 so this transporter has been characterized and which plays important role

in transporting the strictosidine out from the vacol into the cytosol so that strictosidine

subsequently be deglucosylated by the action of stricty in glucosides so this is very important so

and so far only two Transporters were characterized uh in this catharanthus system one

was uh uh ABC transporter an ABC transporter and that

exports catharanthine into cell surface and the second one is basically

a proton driven antio which transports vindoline

and transports let me write vindoline and

Catan into the

vacle for subsequent conversion now and how they have checked the function that is very interesting when they have

characterized this transporter uh they uh this when they have have the gene so they have silenced this so they have

made the silence ing that means silencing of rather I will write implant silencing of

CR n PF 2.9 and then what will happen so that

will be very clear if I draw two simple chromatogram to explain so one for example is your uh

control so where this is basically the LC Ms

chronogram CMS ion chromatogram

sorry CMS chromatogram

showing strictosidine so this is the control so the control means that this is

bigs with empty vector and the one which

is the real one so that means the silenced one so that will show so this is now if I highlight with the

red one then it will be clear so this peak is basically for the strictosidine

which is present in

vs silenced plant whereas in the wild type plant or

the control plant strictosidine is not present because strictosidine will immediately converted into strictosidine

aglycone so this is the way to characterize once you have made the function disrupted so strictosidine

cannot be transported out and therefore if extract is made and run in lcms idin can be detected so this is a very unique

work and and it's a very significant discovery that is why this was published by Pine

atol and the group leader is of course Sara e o conod and that time she was in JN Center

UK and this paper published in the famous Journal nature plants in the year of

2017 volume 3 article number 16208 so with this I have given you more or less the overview of the

compartmentation and uh right and the role of Transporter uh which plays important

role in transporting this metabolites so with this I end this class thank you very much