Comprehensive Overview of Benzenoid Biosynthesis and Volatile Emission in Plants

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course on pharmacognosy and metabolic engineering this is lecture number 57 where under at the broad domain of

phenolic I will now discuss about the biosynthesis of volatile meno uh in the previous class we talked

about volatile phenolics so here mostly Focus will be on the benzenoids that is C6 C1 metabolites so the concepts to be

covered the origin of benzenoids from Phile propanoid volatile benzenoids that is floral Cent the plant species claria

and ponia these was used as a model plant and what has been discovered uh with this plant I will try to present

then after that I will talk about the benzenoid network in plants and then once we discuss all these things then I

will discuss little bit of the work what uh my La is doing on the benzenoid volatiles with a very popular and Indian

floral fragrant flower which is jasam Sak or belly or Bella uh okay and then we'll also talk about

the emission mechanism of this benzenoid volatile emission particularly in terms of physiology so hope you will find it

interesting as well so again the point is that the simple phenolic C6 C3

okay uh so this is so benzenoid means it will become C6

C1 so that will be our point of discussion so

uh uh this is basically one paper uh which published okay there is a mistake here I

must correct it so uh this is a paper published in uh plant physiology not in uh plant cell

environment I think this is volume numbers are fine only the name

of the journal anyway uh now I have corrected it so what we see here is basically the uh trans camic acid

starting point of the uh Phile propanoid pathway and which makes parakum aric acid by cinate 4 hydroxy

which should be here and then uh minded this is the year of 2000 so I have picked up intentionally this paper so

where uh it was proposed that from parakum aric Acid Eugen is formed by multiple step reaction at that point of

time it was not clear uh so how it is formed but now we know that basically

the fural KO basically the fural KO contributes towards the formation of monolignol pathway leading to Conifer

alcohol and that makes ugal whereas parakum alcohol also contributes to chavicol so the point is this at that

point time Point uh the eugenol to methy eugenol that is called ISO ugol methy transference that has been

characterized and bit as I said that is the first enzyme discovered later the BH name was given but B was discovered in

this context with the plant species called claria

brii on agressive family which produces a big flower which emit both benzenoid and tarpo volatiles so when they

analyzed the benzenoid volatiles they found that that was dominated by benile acetate along with that methy eugenol

methy salicylate methy benzoate so these are the components which are dominated in the uh sand bucket of claria

breary flower so once they have identified this compound by gcms analysis they try to

work out how this is formed and accordingly what was clear at this was the methy so there must be a methylation

reaction similar like like methy isol was detected which must have produced from ISO ugol so isol methy transfer

will be there similarly eugenol will convert to methy eugenol by a similar enzyme and benile acetate in the context

from benile acetate the beat is a trolan Noel enzyme was discovered which converts benzy alcohol to benzy acetate

and we know that in this conversion it requires atile qu similarly methy salicylate was also discovered as a one

of the volatile components so in this context salicylic acid methy transparence or um or it is later named

as bsmt benile salic methy transparence has been

characterized and methy benzo was also found so the benzoic acid methy transference was also

subsequently characterized so I mean here that BTT bat iemt all these enzymes and subsequently genes were

characterized from claria flowers and now what we will see here is this that

the salicylic acid carboxy mile transfer is uh which I saidt the name is like this so this what

you see that the the one is the D one uh that is the petal time post enthesis d one that means the when the flower B

opens fully Bloom so and minus one is the one day before minus 2 is 2 days before so what you see see that uh

the maximum activity was found just one day before particularly for the smt activity

and then once it opens the activity declines okay and then it further declines uh where but in case of bit it

is a different Trend so the flower opens at one that is one day time post anthesis and it

continuously enhances its activity leading to the formation of benile acetate so that means that as long as

the flow Alles for 3 4 days it continuously emit benile acetate and there is maybe a relationship between

benile acetate and pollinator attraction that is another aspect and for but in case of uh isal methy transfer as you

see the maximum activity was found here at day one so that means this enzyme the physiology of this enzyme

activity differs from enzyme to enzyme and that means how the compos the the composition differs with time at at

certain point of time it may be more just at the time of opening it may be more of uh your um methy eugenol or

isomethyl eugenol and then that level moral maintained but what happens but the

smt which was maximum just before the day of opening with time it releases down so there are physiological

significance but that is not our point of discussion next what we see this one uh Discovery uh in

2006 where where they have characterized uh just a year before they characterized bpb or BBT I have

mentioned in the previous class that this requires benzo Co

and and uh benile alcohol which joins this again a BHD family enzyme which is

bpb and it makes benzil benzo similarly benzo qu can also joins with

Phile ethanol that is also an alcohol and makes Phile ethy benzoate so uh in 2005 this enzyme was

characterized at the molecular level and what we see in 2006 what they have done they try

to block this activity uh using RNA approach okay and

they want to see what what is going to happen as a result of this blockage and what has been found that when um

elimination of benile benzo formation decrease the endogenous pull up benzoic acid and methy benzoid

so elimination of benzil benzoid means that the benzo benzoid content is getting down why because bpb is blocked

when bpb is blocked then what will happen it it decrease the enous pool of benzoic acid and methy benzo so that is

interesting uh that these two contents are also getting decreased okay so hardli are what has

been thought that benzoic acid is formed through this

Pathway benide to benzoic acid Benz deide is formed from camic Acid okay and then benzoic acid is formed it converted

into methy benzoid but now with blocking of bpb what has been found both ba and methy benzoid has decreased so that

means yeah there must be an alternative root uh for the formation of benoic acid

that means there must be and let me erase this that must be and alternative root for the formation

of benzoic acid from benile benzo so which is yet to be

characterized so this confirms that contribution of benzo benzo towards benzoic acid formation that is entirely

a new hypothesis which is coming out from this work and that is why so next point is this look we what I have said

even in the previous one that uh all is started with phy alanin although filine alanin I have not shown here it is not

shown here but you can add here pH and put uh arrow and put the

enzyme P so phy alanin and and we know how phy alanin is formed phy alanin is formed

from simate arogenate pathway okay now simate arogenate pathway so

philein here is your uh here is your phy alanin and here is the tyosin it comes from arogenate

uh and arogenate is basically coming from prenate and preate is coming from Kismet okay so this is basically a paper

which published in 2010 by the same group where what they have done so they characterize a

arogenate dehydrates so arogenate dehydrates is here

ADT so they block ADT and by blocking this RNA suppression reveals Phile alanin is synthesized by

the arogenate pathway uh okay so let me read the statement that adt1 option in ponia Petal significantly reduce the ADT

activity which is clear and levels of Phile alanin of course phy Alin level will be reduced and the downstream Phile

propon benzenoid volatiles that means the benzenoid volatiles also will be reduced

and but unexpectedly arogenate levels were unaltered while simate and uh tryptophan

uh level were decreased so tryptophan level decreased cic acid level decreased but arogenate level

remain unaltered which is difficult to explain but feeding with simate led to the

formation of prenate and Phile pyrovate accumulation that means when simic acid feeding was

done so that leads to formation of both the things both both Phile pyate as well as prenate and

that leads to partial recovery of Phile alanin which suggests that this may be this may be another alternative root

of phy alanin formation although the main roote of finy alanin formation is this root that is

from simate corate prenate arogenate phine but there may be another

root involving from prefet to Phile pyit can contribute to the uh phy alanin and another important point is this the

regulation lies Upstream of phy alanin that means the volatile emission in ponia is basically regulation lies

Upstream of finin that is also very interesting so it's far ahead of this so another uh we see another interesting

work here in that published in nature chemical biology in the next year where they have characterized prefet aminot

transfer so prefet aminot transfer is basically a it was not characterized before and

that was basically upon this characterization what scientists claim that they have now completely understand

the pathway of py formation from the uh dhp starting with the dhp synthes so prenate aminot transference directs the

carbon flux from prenate towards arogenate and the which is predominated in the phy

Anin biosynthesis so that is interesting and what they have done they try to block these activities and as a result

of this they found that it substantially uh reduce the uh benzenoid volatiles levels in the p

flower so we started talking about benzenoids benile benzoid there are two benzen ring but it's is it's all started

with C6 C1 so this is benzy benzonate and this is Phile ethy benzo so you can also write PB it's

fine so uh and the bpb enzyme is basically either joining penile ethanol with benzy KO or it

joins enil KO with benile alcohol and it's making benile benzoid okay so this is

well characterized and and also bsmt is well characterized uh and benzal deide benzal

deide to benzoic acid which is simply a aldhy de hydrogenous which has also been characterized okay and uh camet to

Camile qu uh which is also uh characterized uh or Kumar sorry

this is karic acid karic acid to Kumar KO which is by a for enzyme which is well characterized and uh this pathway

eventually leads to isol or ugol that we have seen in the previous class so that's fine but the point here um the

networking says that from this karic acid to the benzal deide formation that means karic acid is C6 C3 this is

C6 C3 to when it comes to benzal deide benzal deide which is C6 C1 all this c61 so this is a chain shortening so there

may be three different ways these things can happen Okay one is called non beta oxidative root uh or non oxidative root

where there is no involvement of as such the qu Harvester okay so where straight away

karic acid can form benzal deide by through the formation of one unstable intermediate which is three hydroxide

three Phile propionic acid this one intermediate and it makes benzal deide and benzal deide subsequently converted

to benzoic acid benzoic acid subsequently converted into methy benzo and what we have learned in the previous

slide that benzy benzo also contributes to benile Alco benzoic acid but this has not yet been fully characterized now the

other point is that one is this is non oxidative and now the other one is the core beta oxidative pathway so here a

scientist put lot of effort and finally they made this pathway confirmed so first enzyme what they have

characterized which is called CNL which converts this camic acid this is camic acid

two okay I made I made a slight mistake so this is basically camil KO this is not Kumar because Camile KO St forms to

benzal deide not kumaro so kumaro will form the four hydroxy benzal deide so this is camil so this is camil qu this

is camic acid this is camil KO so Camile qu will enter into the paroxysm and then there is an enzyme called CNL which is

similar to 4cl which is Cam qu lias which converts cam acid to cam qu and then this camil qu

subsequently uh forms through a beta oxidative pathway benzo qu so that is three hydroxy three Phile propano qu and

then then 3 o means three oo3 Phile propano KO and then it makes uh benzo KO and what has been

found with time that this is very unstable compound so camil qu can form to three oxy three

Camile three oxy three Phile propanal CO as a single step and in the next slide we will see what is the enzyme but at

that point of time when they have published this paper in 2012 so they have characterized this K

which is ketoy so ketoy is basically doing the uh reaction which converts three Oxo three Phile propano KO into

benzo KO so this has been characterized in this work so and this is what happens let us look inside the peroxisome so

camic acid enters through some Transporters which is not clear then core beta oxidative pathway is is

operating which makes benzo qu so the C6 C3 now becomes C6 C1 okay then from benzo KO it makes benzoic acid T stands

for theris so the KO will be broken down so benzoic acid is formed this benzoic acid may come out into the cytool and

converted into benzo Co so here is a bzl this is benile coil I guess or or there maybe a

qu Li is exist here which straight away convert benile qu to benzoic acid to benzo qu without the involvement any

transporter so this is the possible root of benzoic acid trafficking the only point is this the benzoic acid is a very

simple molecule okay so this is benzoic acid but you see that its formation is so

comp FX okay uh here in the past okay in 2009 so the

ketoy has been characterized as I have mentioned which converts three Oxo three Phile propano Co which is a C6 C3

compound into benzo KO which is C6 C1 so here the chain shortening occurs so this is C3 chain is now becoming C1 chin so

this this was well characterized and that was published in uh pns in 2009 okay so next one

is uh bpb we have discussed we will not discuss now come to more or less this one so

where uh they mention here is that they confirm the presence of a cam Co lies which converts camic acid into Camile Co

but this Camile KO lias is basically stays inside the parm okay

and but also it is there is a possibility that a non oxidative root is also functioning as the camic acid

converted into B non o bya non oxidative root to benzal deide which I have mentioned and this benzal to benzoic

acid then methy benzoid is all formed so here basically they are confirming the CNL or the cinam KO lies plays an

important role leading to the formation of benzo KO and all for Cals are very important

Co liases are very important so basically they play with that and see that how blocking foral really affect

the pathway and then finally why uh uh this is in plant cell environment published in 2014 now this is the clear

picture so what we see here is camic acid is in the cytool that moves inside uh and then CNL enzyme is there which

converts the camic acid into camil qu and then there is a CHD so this is a Camile KO hydrates dehydrogen so which

converts Camile KO into to three oo Phile propano qu so in between there is an intermediate I say three hydroxy 3

Phile propano W but that is an intermediate so this basically enzyme catalyzes uh uh uh this two reactions

but for the Simplicity cile qu if you incubate cile qu with this enzyme you will straight away find three3 PP qu and

this uh this subsequently by a ketoy make benzil KO so here is the chain shortening occurs this is the chain

shortening enzyme here and then this benzil KO will move out and utilize for formation of Phile ethy benzo or benzy

benzo as the case or it may also uh leaving the KO out can form the benzoic acid and benzoic acid can subsequently

utilize for methy benzo uh and so on similarly the camic acid can convert it into parakum aric acid and parakum aric

acid to this ultimately it forms uh Conifer alcohol or it and uh through multiple

root so which is the monol liol root so that we are not discussing here but then cfat I have discussed in the last class

Conifer alcohol atile transfer is and then which makes Conifer acetate this is conifer acetate and then there eugenol

synthes will make eugenol isol synthes will make isol and then a subsequent methy transference will'll make either

methy ugal or methy isome methy ugal as the case may be so this is in in brief the latest update of the

benzoic acid biosynthesis again as I said the benzoic acid is a very small molecule very small molecule but this

benzoic acid formation requires such a complex route like you remember the monal pathway so when we introduced the

metah hydroxy so formation of Cafe from Kumar simply it requires at three position o but it

is not that easy it involves joining with either Kumar or uh joining with either quinet

or uh simate and then there will be cleage uh and then uh then it then there will be Hydrox silation it forms the

subsequent Capo MO and then subsequent conversion so that means apparently the structure looks very simple so

chemically possible to make this to this but in the biological system it's uh pretty complicated so with this I now

move into uh the work what we have done in past years regarding the uh s volatiles emitted by jasminum species so

jasminum species like I mentioned belly or Bella then there is another called ji or Jui so the Bell is jasminium sambuk

and the Jui is the jasminium oriculum so this is a holistic schematic diagram what what

is showing here that uh the volatile s volatile consist of both tarps and benzenoids so in this class we are

focusing on the benzenoids because tarpo aspect I have covered in previous classes so what is showing here that

multiple cellular organis play important role in the synthesis and storage of the volatiles so it's stored sometimes at

glycosylated volatiles that means it's it under it joins with the glucose molecule and as and when required there

will be deglycosylation which is by the glycosidase enzymes and as a result of that this will be emitted and

the emited volatiles will subsequently released okay so this is a general so now I see that the when you work with

jasam sok so jasam sok actually um it it it it remains alive for more than 24 hours so we we saw how the flower

changes starting from the bar to the full opening until sence how it changes uh how it openings the petals and each

petals have multiple holes two or three layers of petals and how uh

the changes occur throughout the floral lifespan is basically showing here and then what we have seen here look the

benile acetate now you aware about benile acetate is found in uh claria bori so from there the be8 has been

characterized and the also uh in petunia so now here also in jasam sambuk we also detected a huge amount of benile acetate

and basically then we detected in the flower how the benile acetate uh content changes with time and this shaded

portion are is the night time because it's a white flower it emit benzenoids so benzenoids are usually emitted at

night time which attracts The Moth so all all these things that maximum emission occurs in the night time and

that is dominated by the menzy acetate then methy benzo then methy Sil this is the typical flower of jasminum

sambuk so now not all benile acetate is emitted so or even benile if benile acetate is

emitted but benile alcohol it prefer to stay as internal pool so it is not emitting but it is staying there so we

also measured that what is the content of this internal pool of volatile compounds throughout the floral lifespan

so uh it's like this so a a substantial amount of benile acetate also accumulates as internal pool later it is

emitted out so so this benile acetate May store as uh unconjugated volatile and later it emitted or it may undergo

glycosilation also okay so this is another simplified photographs of jum some

flower stage wise so S1 S2 S3 S4 S5 S6 so that is that uh stage one is at 2 p.m. day 1 uh when the body is still

growing and then H2 is basically uh the 8:00 p.m. and then H3 is basically uh 2: a.m. at the midnight

when is about to open and then S4 stage at 4:00 a.m. 8:00 a.m. in the morning when it is opening and then S5 is this

fully opened and then A6 that is now it's it it is ready to undergo cence so at

these six different stages uh we studied so this is one more thing that the as I said benile alcohol

it it it it undergo glycosilation so a glucose m is added and then it store in the floral tissue particularly in the

vac and then upon requirement the glycosilation occur by enzymatic reaction leading to the emission of pre

endogenous volatiles and uh at that for at that six stages that means the stages are here S5 and S

okay so the the Phile alanin ammonia lies because that is the pal enzyme which is

the starting point for this benzenoids so where it remains maximum it remains maximum uh just at the time of opening

of the flower at S3 stage okay and whereas the the beat activity beat activity uh detectable beat activity is

there although maximum beat activity occurred at the S4 stage that means at this stage maximum beit

activity or maximum pal activity is here okay pal activity first is required because pal will come first it makes

camic acid and then later the bit will make the subsequent uh benile acetate so that that makes sense so more P activity

is the earlier stage and be beat activity will be the later stage and this is basically the glucosides

activity that is more or less constant throughout the uh stages of flal lifespan now this is interesting you see

that uh I will only show the pal so this is Knight so pal activity reaches its maximum here uh at uh 12:00 midnight or

so and then it it's continuously it's coming down again in the next day evening it's slightly reaching up so

that is important because pal activity and Then followed by the other reaction leading to the formation of the Phile

propanoid pathway and this is the beat activity beat activity but it it it it reaches maximum uh much later than that

of the P activity uh and then benzoic acid methy transference

which converts benzoic acid to methy Beno that has also been measured throughout the floral lifespan and here

you see the maximum activity during the night time is the night time whereas salicylic acid me trans transport is

maximum activity occur in the daytime and uh that is uh but during the B condition so I I have shown that

salicylic acid methy transfer is more during the B time so if you see in in case of the

previous slide if I remember correctly here also maximum salicylic acid methy transfer is

occurred at minus one that is before the opening a similar thing also we observed in our case as

well this is the time petal opening it and this is before so and this is basically a nutshell about the major

volatile compounds emitted by the jasminum sambok flower and also we have uh characterized partially that a

transcription Factor like odorant odorant plays important role so odorant expression basically correlates with the

expression of benzenoids so we have partially characterize the autodent transcription factor and finally based

on the different uh study like volatile emission enzyme activity like gene

expression then a holistic mechanism was presented here what happens in the cell so that that

the vol compounds are synthesized in the cell they stored in the vacle later they release the the free volatiles internal

pool okay and then they released as the emited volatile so there is a role of Transporters because we also

characterize a few transported genes uh we have studied the expression based on the transcriptomic information available

so and uh so this is basically shell what happens in the jasminium sok flow leading to the emission of volatile s

volatile which dominated by benzenoids but also it's it it also constitutes uh tarpo component as well

dominated by linalol so this is in general the outline of mechanism governing s volatile synthesis storage

and emission in jasminum samb uh so don't get daunted it looks pretty heavy but this is just to give you an idea

about how reaction happens so and that leads to sent so the scent means what you is what you get it in

your all Factory Nar through nose so it's not a single component so it may be starting from 20 to 60 70 components

that all constitute that specific scent so that is the message so with this I end this class thank

you