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Module 1: Plant Metabolism

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Secondary Metabolites and Plant Commerce

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So, we were talking about Secondary Metabolites; so, only about their catabolism we also spokeabout that secondary metabolites they generally have turnover processes. The rate of synthesisalso depends on their rate of catabolism or metabolism which means which may include therate at which they are getting transported to the storage sites or it may also includethe rate at which they are getting bio-transforms for detoxification and storage subsequentlyor directly being used in pathogen attack. So, the rate of synthesis is also dependenton the rate of metabolism of the secondary metabolites.So, in its interaction with the environment a plant must always be capable of using itsecondary metabolites. So, generally, we have spoken about this that at the site of storagemay not be the same as the site of synthesis. So, it is transported generally through xylemto the parts where it has to be stored and we know for example, what are the differentways in which the plant stores it. It may store it in vacuole, it may store it inintracellularspaces or tricombs or glandular structures, hairs, where it is stored or epidermis orcuticle where it is exposed and where it is needed for the line of defence.Now, protective mechanisms have been involved in order for the plant to protect itself fromthe toxic effect of the secondary metabolite. So, what kind of protective mechanism didwe talk about? One is biotransformation, like glucose moiety addition glycosylation. Whatelse? The storage and vacuoles. Or keeping the final step where the precursor will getconverted to the toxic metabolite, only once the cell is damaged. So, which means the enzymesof the final step are kept in the vicinity of the precursor moiety. So, transport. Yeah.Ma'am. So, if the transport is of xylems only you need a direction. So, if the secondarymetabolites is produced at somewhere at the top. So, does it flow phloem also takes partin the transport? So, it generally depends now. When we saythat at the top, at the top is very ambiguous. Now top is everywhere above the ground level,so it is not that it will only happened at one single point of the plant, it will happenedaerial. So, we can generally bifurcate as aerial or underground parts. So, it is a veryambiguous term to even answer that top would mean what.So, transport and there are active as well as passive processes. So, active means againstthe gradient also it can flow. Now, transport mechanisms are passive and active processesare involved in transport from the site of synthesis to storage. Now, when active processesare involved which means some transport proteins would be involved. Now, upon completion ofall possible chemical transformations they are re secreted in the surrounding mediumwhich is your xylem or the cell sap. The secondary products are sometimes transferredvia xylem to various parts of the plant. Now, if you remember we were talking about inducedsystemic resistance and SAR, Systemic Acquired Resistance, in both the signalling moleculeswere transferred through phloem dependent signal not the xylem. So, I if I think bothare involved in the entire process in the signal cascade or the pr proteins flow todifferent directions or even your for storage of the secondary metabolites transport.Now, diurinal cycles and annual rhythms are involved in the secondary metabolites synthesisand when we say diurinal cycles which means that they undergo both temporal and spatialvariation. Now, this is temporal which means that the synthesis may be following24 hourcycle or it may be following an annual rhythm, once in a year, twice in a year. So, the synthesisexplains. Maybe, if it is playing a role in the developmental stage of the plant thenit generally follows the cycles, patterns. Now, these variations are caused by the variationsin temperature or light conditions in the course of the day. So, how do you think thatthe variations which are following a particular rhythm the plant is able to maintain theserhythms? By manipulating what? How is the plant managing this? Think logically, do notread. Where is the control? Synthesizing the proteins only when like dependentreactions . Now, everywhere now these are enzymatic biosyntheticpathways, enzymes are involved, enzymes are further controlled by the proteins by transcriptionfactors. So, there are different modes of controls with the plant, the metabolism iscontrolled. Now, by controlling the metabolism ultimately affecting either the activity ofthe enzymes or the expression of the enzymes, the plant has the handled to manipulate therhythms. Now, generally, in plants you will see that enzyme activity can be impacted byyou know what are the common factors. What are the common factors by which you thinkthe enzyme activities can be can get affected? You must have already learned it.It is actually pH. pH, temperature. What else?Light. Light can impact.Now, cells in a tissue are exposed to signals from the surrounding tissue. So, endogenouschemical gradients can be experimentally induced in cultures for differentiated cells. So,generally in under in vitro conditions when we try to do optimizations for maximum productionlike for example, we add elicitors, which means stress or we manipulate ph temperaturerpm. So, ultimately it is impacting what? What do we check? If you want to dig downthe mechanism when we add specially the elicitors yield enhancement mechanism which means perunit bio mass the synthetic capacity of the cell, then we look for the rate controllingsteps in the biosynthetic pathway. We see that what is the impact of this particularparameter on any of the rate controlling enzymes, whether on their expression or on their activitylevels. Now, the possibility of decoupling productionand storage capacity. Yesterday, we were talking about this. Generally, it has been observedthat these are all intracellular, generally, these are intracellular compounds, the cellproduces inside. Now, in order to improve the production rates if you will increasethe rate at which it is metabolized or transported outside will increase the rate of forwardreaction which is the rate of the synthesis. So, either under in vitro conditions you havepresence to absorb the product or you decouple the production and the transport to the surroundingmedium or you add permeabilizing agents, which can reversibly permeabilize the cell for theproduct to continuously keep coming out as it is getting synthesized.So, these are all among the strategies which we will be discussing which can improve theproductivity of the secondary metabolite in plant cell based bio processes. Now, the syntheticcapacity of dedifferentiated tissue, we know this. Often differs substantially from thefully differentiated tissue. We saw that we were discussing that secondary metabolizemetabolismin plants is a function of cell differentiation sometimes morphogenesis, organogenesis.So, now, it is connected to higher order functions which involves cell differentiation. Now,therefore, under in vitro conditions you would find that the cells in a callus may not beas productive as if you convert those cells into organogenesis or more differentiatedor if you see that the callus is more compact there is more strong cell to cell contactthen the secondary metabolite biosynthesis sometimes gets enhanced.Now, see these are the kind of balancing acts between, now if the callus or the cell speciesis aggregating which is which may be good for the yield of the secondary metabolite,but that can be a limitation for, we are improving yield by making it much closer coming in contactwhich would improve organogenesis biochemical, signalling between cell to cell contact wouldimprove, cell differentiation might the cell differentiation signals might get inducedwhich is good for secondary metabolite biosynthesis. But where is the limitation?mass transfer limitation. Mass transfer limitations. So, the scale upwould ultimately get affected, the mixing will get affected which may cause oxygen gradients.So, it is always balancing between the merits and the demerits of any particular strategy.Now, production of secondary compounds is usually most efficient in tissues farthestfrom the meristematic activity. So, for higher biomass productivity, you would need thatthe cells continuously keep dividing, and for secondary metabolite biosynthesis, theword secondary itself says that resting cells would produce better. So, it is a non-growthassociated production. So, therefore, decoupling of the production phase and the growth phaseis sometimes found to be better in secondary improving the secondary metabolite biosynthesis.So, for example, like in callus most of the cells will be parenchymatous in nature. Now,you will see that once the cell are metastatic, they are still expanding growing, multiplyingand once the cell reaches a matured state then higher order functions or biochemicalactivity would appear. So, therefore, it has to be seen that, but for inducing callus,for inducing cultures you need young explants where there is higher metastatic activity.But production of secondary metabolites you it would be better in matured state of theculture. Now, some of the examples. Expression of hyoscyamine6 beta hydroxylase RNA occurs only in developmentally young roots. You will observe that some ofthe secondary metabolites uh appear only once the shoot gets up or is induced or the rootsare induced, still it is in callus or cell suspension phase, the secondary metabolismis not induced. There is no secondary metabolite observed.Formation of small roots in cell aggregates of coleus species induces synthesis of forskolinor diterpenoid which is of commercial value. Similarly, major components of onion volatileoils are only formed following shoot induction. The onset of cardiac glycosides synthesisin cell cultures of digitalists has been linked to shoot induction. So, these are some ofthe examples which demonstrate that organogenesis is linked with your secondary, might get linkedwith secondary metabolism. Nicotiana tabacum callus cultures, root induction is only inducedby the addition of nicotine. So, it is two way. So, nicotine you add and then you seethat the; it has induced what? Root induction or yeah, organogenesis in the cultures.Now, plastic metamorphosis also sometimes has been observed to be linked with secondarymetabolism, where they have observed inlycopine biosynthesis. Where, adding a herbicide intomato cell cultures as a it upon induction of lycopine biosynthesis by the herbicidewhat was what was observed was that the chloroplast started diminishing and they started convertinginto chromoplasts that red color pigment started appearing. So, in nature what might be happeningthat as the lycopines synthesis has to happen for the red thing to come up the tomato orthe fruiting or the maturity of the fruit to happen the chloroplasts start getting diminished,which means at the proplasted stage the pro plasted rather than getting converted to chloroplaststart getting converted to chromoplast. Chromoplast.Now, developmental of chloroplast has a or sometimes it is the other way round. Likewe are observing an alpha tocopherol as well that the more green is the callus, the betteris the alpha tocopherol yield. So, it is linked to chlorophyll biosynthesis, even in the biosyntheticpathway there is a link between chlorophyll bio synthesis and vitamin biosynthetic pathway.So, similarly accumulation of cardenolides. So, it is morphogenesis effect an example.Accumulation of cardenolides in digitalis cultures has happened only after the initiationof the green tissues or adventitious embryos. We have observed in viola odorata also thatthe cyclotide array or the cyclotide biosynthesis is stable in an organ culture like somaticembryos. Callus to begin with when you induce, they show that the cyclotides arrays therewhen it is a fresh callus, but with subsequent years of sub culturing the biosynthetic capacityis lost. So, this is an example which shows us that organogenesis is linked with the.Now, the same callus if you converted it into somatic embryos the cyclotide array is revived.So, these are some of the examples of plant products of commercial interest where theray it ranges from pharmaceuticals, then food colors, flavors, fragrances, sweeteners, artificialsweeteners and agriculture chemicals, like pesticides or even fertilizers.Now, some of the commercial productions which are happening for the phytochemicals usingplant cell based bioprocesses are these. Your anthocyanins, betacyanins, so these can bepigments coloring agents, flavoring agents. So, there are different kinds. Betacyanins,carthamin, geronoil, ginseng health based for good health. Shikonin, it is a coloringagent. Cocoa cells, bilberry cells, so these are different kinds of products which havebeen commercialized by Japanese farms, by German farms, by US, by Israelis. So, theyare already there in place are being used for what all uses? If you can see the tableas textiles, as coloring agents, as pigments, as dietary supplements for as nutraceuticals,so as cosmetic agents. These are pharmaceuticals which are gettingproduced commercially. Anti-cancer, anti-biotic, anti-inflammatory, then for the treatmentof motion sickness, nausea. So, then pharmaceuticals, pharmaceuticals like berberine, then paclitaxelis well known it has the taxol drug which is used for anti-cancer, vincristine, vinblastine,then even your podophyllotoxin. Now, heterologous proteins. Heterologous proteinsnow the interesting part is it is not only now the plant cells are also replacing CHOsin some cases where production platforms. Now, CHOs are increasingly used as for productionof recombinant proteins, human proteins as vaccines or or for animal vaccines or evenas anti-bodies, bio-similars is one big area, so where CHO cells are used.Now, there are examples now where some are in human trials, but some plant based theserecombinantproteins which have been reduced plant using plants cell based bioprocesses are now approvedby FDA and are now marketed. So, some of the examples are your Gauchers disease, a shirebios. It is being produced using carrot cells suspensions got approved by FDA in 2015 andit is now marketed it with protalix, it is an Israeli based company which first madeand now it is being sold. Then vaccine against Newcastle disease virus. It was being producedusing tobacco cell suspension culture and it is marketed by Dow Agrosciences. And similarly,there are many others. So, this is just to show you people that thereis so much of scope and the world is gearing up to and why do you think that it is on therise. So, one is it is a green technology. So, immune response against the plants thechances are less. So, it is a sustainable greener, environmentally, friendly, technology.So, please remember yield now on would be referring to y p by x which means the amountof product per unit biomass, and productivity would be volumetric productivity which isthe product tighter. Product tighter is product? Concentration.Concentration per unit time. So, we will be working around in all these strategies eitherit will be impacting both the things the product yield and the productivity or it will be impactingoverall productivity through by improvement in; if it is not impacting product yield,but still the productivity is high can be because of, time. So, from where is that coming?Probably, generally these are intracellular products. So, these are generally intracellularproducts. So, when we say product tighter what does it mean? This will include whattwo parameters? Amount and volume.So, from where is this amount and volume coming? When we say productivity in an intracellularbased product it will be suppose grams per liter per time. Now, this gram per litterper time is a club of what two things? Ok.How do you measure gram per litter per time? How would you measure? Think that ways.After all the likes amount of in total volume before the extraction. No. From the totalvolume. So, how do you get the total amount of product?From different. Concentration.What concentration? Think a little math is involved. How would you calculate? Start fromthe scratch. I said product productivity would be; assuming it is an intracellular product,so she is said it will be extracting from the biomass. So, how will you calculate? So,how will you get two grams per litter per unit time?Total amount of biomass which we had initially iswill come in denominator and after all theextraction steps ah. So, for extraction every time you will havethe entire bio mass. No.For extraction? No, no.How, how will you do it at a experimental level? Think that ways. You are running ashake flask or you are running a reactor, what would you do? You will every time harvestthe entire bio mass and then you will dry it, then you will extract using the entirebio mass? No, that is thisif if in 1 ml I have 1 mg.Ok. That is 1 mg per ml to what would be therein 1 litter. So, it will be. Right. So, here 1 ml would be there, ml iswhat then? ml is cell know cell bio mass, the total mass.Will it be volume, cell biomass? Ok. It will be by weight by weight.Weight by weight. Now, this weight by weight is called what?Yield. Yield.Yield So, one is yield.Ok. The other would be?Rate of productions. Rate of what?The reaction time . The mu.So, this mu is specifies what? The kinetics of the.Of? bio mass.So, now it will take care of your. Now, this was if you think mathematically it is gramper gram bio mass multiplied by the concentration of the biomass. So, this will give you theproduct tighter, is not it. And then the time in which that product concentration of thebiomass is achieved. So, that is what is s overall volumetric productivity. When youhave mu it becomes? It becomes? .What does it called when you have mu? What will it be called?Cells . Along with the yield what will it be called?. Think about it this.. It is simple maths. I want you to representyour product after evaluation as the volumetric productivity or the product concentration.How would you determine? Just think on those lines, then you will be able to figure outthat what should be your two important responses when you are optimizing a process. What willbe your two objective functions? Ok. So, we will continue tomorrow.