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heat How to make the hottest peppers - if you know a good plant geneticist

nice work, pepperhead!
In the last part, it basically says that a disabling mutation of Pun1 (required for capsaicinoid synthesis) has given rise to the recessive gene pun1, which is the heatless version.
 
What I find odd is their suggestion that humans have been selectively breeding peppers for less heat ("...pun1, used in pepper breeding for nearly 50 000 years...") since the start of human "behavioral modernity." I wonder what evidence they have that we have always wanted less spicy peppers?
 
Pepperhead said:
I'm no scientist, but I tried google and wikipedia to translate this (to me) garbledygook.  I'm sure there are mistakes, and I'm sure it's oversimplified, but I tried as much as I could to put this in layman's terms.  Corrections are always encouraged. 
 
Everything in red is my attempt to translate what I was reading.
 
[SIZE=12pt]Pungency in Capsicum fruits is due to the accumulation of the alkaloid capsaicin and its analogs. Peppers are spicy because of capsaicin and capsaicin like chemicals. The biosynthesis of capsaicin is restricted to the genus Capsicum and results from the acylation of an aromatic moiety, vanillylamine, by a branched-chain fatty acid.  Only peppers within the genus capsicum produce capsaicin and does it by introducing an acyl radical called vanillylamine by a branch chained fatty acid.[/SIZE] Many of the enzymes involved in capsaicin biosynthesis are not well characterized and the regulation of the pathway is not fully understood. We are still not sure how the enzymes are involved in making capsaicin. Based on the current pathway model, candidate genes were identified in public databases and the literature, and genetically mapped. We genetically mapped certain genes we already knew about. A published EST co-localized with the Pun1 locus which is required for the presence of capsaicinoids.  (I think based on the next sentence) We Identified a gene that is required to make capsaicin and capsaicin like chemicals. This gene, AT3, has been isolated and its nucleotide sequence has been determined in an array of genotypes within the genus.  AT3 is the name of the isolated gene and has been found in a lot of types of peppers. AT3 showed significant similarity to acyltransferases in the BAHD superfamily. AT3 does a lot of the same things as a certain family of enzymes called BAHD.  The recessive allele at this locus recessive gene on this species contains a deletion mutation caused by missing genetic material spanning the promoter and first exon the place in the DNA that starts translating the gene and the sequence that helps make proteins of the predicted coding region in every non-pungent accession tested. Transcript and protein expression of AT3 was tissue-specific and developmentally regulated. Only certain tissues were affected by the AT3 gene and it was regulated developmentally. Virus-induced gene silencing of AT3 resulted in a decrease in the accumulation of capsaicinoids, When AT3 was exposed to certain viruses it was shut off and fewer Capsaicinoids were produced a phenotype consistent with pun1. In conclusion, gene mapping, allele sequence data, expression profile and silencing analysis collectively a bunch of tests indicate that the Pun1 locus AT3 gene in pepper encodes a putative acyltransferase accepted enzyme, and the pun1 allele recessive gene, used in pepper breeding for nearly 50 000 years, results from a large deletion mutation caused by a missing piece of genetic material at this locus in this species.
 
So, a gene (AT3) helps to create capsaicin in a similar way as a large family of enzymes. The recessive gene in this species (capsicum) contains a mutation causing less capsaicin to be produced.  AT3 affects certain tissues and as the pepper grows certain amounts of capasaicin is created. Some viruses can shut down capsaicin development at the AT3 level.
 
Oww.  My brain hurts.
 
How close am I?
Pretty close. Excellent job.
 
 
 
Pepperhead said:
I'm no scientist, but I tried google and wikipedia to translate this (to me) garbledygook.  I'm sure there are mistakes, and I'm sure it's oversimplified, but I tried as much as I could to put this in layman's terms.  Corrections are always encouraged. 
 
Everything in red is my attempt to translate what I was reading.
 
[SIZE=12pt]Pungency in Capsicum fruits is due to the accumulation of the alkaloid capsaicin and its analogs. Peppers are spicy because of capsaicin and capsaicin like chemicals. The biosynthesis of capsaicin is restricted to the genus Capsicum and results from the acylation of an aromatic moiety, vanillylamine, by a branched-chain fatty acid.  Only peppers within the genus capsicum produce capsaicin and does it by introducing an acyl radical called vanillylamine by a branch chained fatty acid.[/SIZE] Many of the enzymes involved in capsaicin biosynthesis are not well characterized and the regulation of the pathway is not fully understood. We are still not sure how the enzymes are involved in making capsaicin. Based on the current pathway model, candidate genes were identified in public databases and the literature, and genetically mapped. We genetically mapped certain genes we already knew about. A published EST co-localized with the Pun1 locus which is required for the presence of capsaicinoids.  (I think based on the next sentence) We Identified a gene that is required to make capsaicin and capsaicin like chemicals. This gene, AT3, has been isolated and its nucleotide sequence has been determined in an array of genotypes within the genus.  AT3 is the name of the isolated gene and has been found in a lot of types of peppers. AT3 showed significant similarity to acyltransferases in the BAHD superfamily. AT3 does a lot of the same things as a certain family of enzymes called BAHD.  The recessive allele at this locus recessive gene on this species contains a deletion mutation caused by missing genetic material spanning the promoter and first exon the place in the DNA that starts translating the gene and the sequence that helps make proteins of the predicted coding region in every non-pungent accession tested. Transcript and protein expression of AT3 was tissue-specific and developmentally regulated. Only certain tissues were affected by the AT3 gene and it was regulated developmentally. Virus-induced gene silencing of AT3 resulted in a decrease in the accumulation of capsaicinoids, When AT3 was exposed to certain viruses it was shut off and fewer Capsaicinoids were produced a phenotype consistent with pun1. In conclusion, gene mapping, allele sequence data, expression profile and silencing analysis collectively a bunch of tests indicate that the Pun1 locus AT3 gene in pepper encodes a putative acyltransferase accepted enzyme, and the pun1 allele recessive gene, used in pepper breeding for nearly 50 000 years, results from a large deletion mutation caused by a missing piece of genetic material at this locus in this species.
 
So, a gene (AT3) helps to create capsaicin in a similar way as a large family of enzymes. The recessive gene in this species (capsicum) contains a mutation causing less capsaicin to be produced.  AT3 affects certain tissues and as the pepper grows certain amounts of capasaicin is created. Some viruses can shut down capsaicin development at the AT3 level.
 
Oww.  My brain hurts.
 
How close am I?
Extremely close. Really excellent job, you obviously put a lot of time and effort into that. I`m impressed.
 
Vanillylamine is the aromatic thing that gets acylated. The acyl group comes from branch-chained fatty acids. The enzyme(s) responsible for catalysing this acylation reaction are not well characterized or understood properly. In biology it is extremely common for organisms to make a certain chemical using a pathway involving simple building blocks (like sugars etc), then having a step-wise synthesis route involving numerous intermediate compounds. Each step in the process is catalysed by a specific enzyme. The last step in the biosynthesis of capsaicin is what is mentioned here - acylation of vanillylamine to give capsaicin.
 
The rest you got. The only thing I`d say is that they used virus` they designed that would specifically switch off transcription of the AT3 gene, not just some virus laying around the lab. 
 
Ají hombre said:
nice work, pepperhead!
In the last part, it basically says that a disabling mutation of Pun1 (required for capsaicinoid synthesis) has given rise to the recessive gene pun1, which is the heatless version.
 
What I find odd is their suggestion that humans have been selectively breeding peppers for less heat ("...pun1, used in pepper breeding for nearly 50 000 years...") since the start of human "behavioral modernity." I wonder what evidence they have that we have always wanted less spicy peppers?
I think the race for spicier is relatively recent. Probably tha spicy pain was less desirable in the past.
 
true enough, but 1) selective breeding of plants by humans can't be that ancient, and 2) in the case of Capsicum, the spiciness was really the reason for the plant's usefulness to humans, wasn't it? who would want to eat a half-rotten leg of rat smothered in bell peppers?
 
I'm curious about that. not enough to download the whole article, though. heheh
 
Not that ancient? Isn`t 9500 years ancient? That`s what the archeologists say about humans using peppers in their diet.  Collecting vs selective breeding is certainly something to wonder about, but if 9500 years is collecting, then maybe 8500 years is selecting? 
 
Hi, Nigel.
The abstract you posted gave a figure of 50,000 years, didn't it? that's the number I'm calling into question. We (at least our species of ape) has been living where chiles are for about 9500 years, presuming that the precolumbian distribution of chiles was restricted to the New World. And if we figured out the phytochemistry of peyote by 6000 years ago then chiles would have predated that!
 
In order to suggest 50,000 years, though--that would require that something--probably birds--would have carried chiles over into Africa from the New World, and also that the barely-modern human would have had the skills to engage in some level of planting and selective breeding. Is there evidence for that?
 
Nigel whats your CV?
 
 
Imagine, in the next 2-3 years, Nigel could be reviewing 3mil SHU Superhot :party:
 
NuHot said:
Nigel whats your CV?
 
 
Imagine, in the next 2-3 years, Nigel could be reviewing 3mil SHU Superhot :party:
PhD biochemistry, postdoc training in in cell biology, neurobiology, molecular biology, genetics, 15+ years academic research in cancer biology.
 
+5 handicap at golf while still a scientist, before turning Pro in 2008. Much, much better player now than then. 
If someone can identify the rate-limiting step in a multi-step process, such as capsaicin synthesis in Capsicums, to boost the amount of end product all you need to do is increase the rate of that single step. To do that you could increase how much enzyme is produced that catalyses that step and if you were lucky and the process was relatively simple, you`d get more capsaicin produced. 
 
Nigel said:
All you`d need to do is add more copies of the gene that encodes the rate-limiting enzyme in the metabolic pathway that produces capsaicin. Simple. Maybe start here!
Instead of copying the gene of interest, insert a 35S promoter before it. This will put the gene into maximum production.
 
Ferby said:
Instead of copying the gene of interest, insert a 35S promoter before it. This will put the gene into maximum production.
Anyway to increase the number of copies of the gene product will work, so yes, in theory that would work too. The problem with consitutively active promotors is it`s hard to predict the outcome, especially if there is feedback inhibition at some step or steps in the biosynthestic pathway. 
 
I do know that Pex hydroponics some of his peppers. I wonder if something chemically similar to Lysergic acid diethylamide but with a different chemical structure, is being used as an active ingredient in some of these hydroponic fertilizers. The reason I mentioned this is because, Im exploring doing a small hydroponics system for my peppers. And the competition to grow, bud and produce top psychedelic cannabis is advertised everywhere in these hydroponic shops ive visited. So I wonder if some of the these liquid products that are used in the flower/budding/part b stage have something a little extra blended into the mix. And if there is some truth to this conspiracy, I could see a hot pepper grown hydroponically could potentially have a real opiate affect on the user.
 
Thinking outside the box here.
It would be interesting to extract some of these chinense cap oils from a super hot and try them in a vaporizer pen.
 
NuHot said:
I do know that Pex hydroponics some of his peppers. I wonder if something chemically similar to Lysergic acid diethylamide but with a different chemical structure, is being used as an active ingredient in some of these hydroponic fertilizers. The reason I mentioned this is because, Im exploring doing a small hydroponics system for my peppers. And the competition to grow, bud and produce top psychedelic cannabis is advertised everywhere in these hydroponic shops ive visited. So I wonder if some of the these liquid products that are used in the flower/budding/part b stage have something a little extra blended into the mix. And if there is some truth to this conspiracy, I could see a hot pepper grown hydroponically could potentially have a real opiate affect on the user.
 
Thinking outside the box here.
It would be interesting to extract some of these chinense cap oils from a super hot and try them in a vaporizer pen.
I`ve had one of Pex`s hydroponic Brain Strains. Holy crap, it was hot. Really hot. Even for a Brain Strain is was hot.
 
I`d expect that hyper nutrition of the correct sort would give you peppers that were as hot as the genetics would allow. Assuming all the needs of the plant were met in the right way. I have no idea what that would entail, as I have zero experience with hydroponics. At some point you run into the problem of the genetics of the plant being the limiting factor. 
 
Nigel said:
PhD biochemistry, postdoc training in in cell biology, neurobiology, molecular biology, genetics, 15+ years academic research in cancer biology.
 
+5 handicap at golf while still a scientist, before turning Pro in 2008. Much, much better player now than then. 

If someone can identify the rate-limiting step in a multi-step process, such as capsaicin synthesis in Capsicums, to boost the amount of end product all you need to do is increase the rate of that single step. To do that you could increase how much enzyme is produced that catalyses that step and if you were lucky and the process was relatively simple, you`d get more capsaicin produced. 
 
 
IMO we shouldn't GMO peppers. Traditional breeding has been good enough. They really don't need to be any hotter.
 
Dulac said:
 
 
IMO we shouldn't GMO peppers. Traditional breeding has been good enough. They really don't need to be any hotter.
I agree with you.
 
However, is it GMO if you add back an extra copy of one gene that is already present in all Capsicum sp.? In the strict sense, it obviously is, but in the sense of GMO organisms that are round-up ready, or have BT genes, it isn`t. Where do you stand? 
 
I thought it might be fun to discuss this in light of the insane arms race going on the get peppers up to and past 2 million SHU consistently. 
 
Nigel said:
I agree with you.
 
However, is it GMO if you add back an extra copy of one gene that is already present in all Capsicum sp.? In the strict sense, it obviously is, but in the sense of GMO organisms that are round-up ready, or have BT genes, it isn`t. Where do you stand? 
 
I thought it might be fun to discuss this in light of the insane arms race going on the get peppers up to and past 2 million SHU consistently. 
 
Yeah, I consider it GMO if you add back an extra copy of one gene. Other things can change without our notice by doing this. This happened with the protein in wheat, which may be the reason for the rise in gluten intolerance. I'm not exactly against GMOs, but we don't test them ethically or even properly. I'm more interested in the gene that blocks heat (if we made sure nothing else changed). Then I could share the great flavors I taste in superhots with other people.
 
Edit: I don't think BT genes and roundup ready plants are a good idea for consumption or the environment (only important to me because it's important to humans). There would have to be some serious studies on how it affects humans and the environment before I think it's OK. The other problem we have is with IP laws on crops of this nature. I'm not OK with people owning genes, and I don't care how hard they worked. I don't see why we cannot take crop loss for being on the safe side. We should test the GMO plants and just use traditional breeding and avoid monocropping. As it is now, I'm completely against this practice.
 
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