\u201cFrom the scientific point of view, you\u2019re always looking for novelty, to bring something new to the table,\u201d said Brumos, the first author on the paper. \u201cOur goal was to find new genes that are involved in the production of auxin, a plant hormone. When we identified that the mutation was actually in a well-known gene, we got super depressed. Then we got curious, the mutation is in the same gene: How did it complement the known mutant?\u201d<\/p>\n
First Up, Then Down<\/strong><\/h3>\nBrumos and senior author Anna Stepanova<\/a>, an associate professor of plant and microbial biology, started with Arabidopsis thaliana<\/em>, the widely-used (and fast-growing) plant species favored by plant biologists. They generated a massive collection of seeds with random mutations and then looked at 10-day-old seedlings for specific traits. Specifically, seedlings with curly cotyledons, the first two \u201cleaves\u201d of a plant shoot, or short, stumpy roots.<\/p>\nThey kept the seedlings with these traits, transferred them to soil, grew them up and bred with themselves. Next, they crossed these \u201cpure bred\u201d mutants with pure bred plants with known mutations in well-studied genes involved in making the plant growth hormone auxin.<\/p>\n![\"When](\"https:\/\/cals.ncsu.edu\/wp-content\/uploads\/2020\/06\/Rooty2-460x355.jpg\")
When a plant with an unknown mutation (TG8B) was crossbred with a plant with a known mutation (RTY1) in an enzyme involved in making a plant growth hormone, the offspring looked normal, suggesting the new mutant complemented the known mutant.<\/figcaption><\/figure>\n\u201cAs expected, when we crossed them, most of them looked like the parents, so we knew the new mutation was in the same gene as the known mutation,\u201d Brumos said. \u201cWe could discard those mutants because the gene was already known.\u201d<\/p>\n
However, there were some normal-looking seedlings from the crosses, which indicated that the new mutant complemented the known mutant.<\/p>\n
\u201cGenetics 101 suggested that the mutations were in two different genes,\u201d Brumos said. \u201cWe got super excited at that point. We thought we\u2019d found a new gene that was involved in auxin synthesis.\u201d<\/p>\n
However, when they mapped and sequenced the new mutation, they were disappointed to learn that the mutation was actually in the well-studied gene, ROOTY<\/em>.<\/p>\nAfter they recovered from their disappointment, the team began to get curious as to how two mutations in one enzyme could complement each other, as the findings were rather unexpected.<\/p>\n
Many enzymes, including ROOTY, the protein produced by the ROOTY<\/em> gene, work by pairing up with another copy of the protein to form a functional unit \u2014 like two halves of a pair of scissors. The team, with collaborator Douglas Grubb, a group leader at the Leibniz Institute for Plant Biochemistry, had a hypothesis that when a copy of the enzyme with the new mutation paired up with a copy of the enzyme with the well-known mutation, the mis-matched mutant pair was functional. Jose Alonso<\/a>, William Neal Reynolds professor of plant and microbial biology and a long-term collaborator of Stepanova, proposed that the new mutation could disable a different section, or domain, of the enzyme than the well-known mutation.<\/p>\nTo test this hypothesis, the researchers teamed up with Ben Bobay, a senior research associate at Duke University, to make a computational 3D model of the ROOTY enzyme \u2014 by comparing the normal enzyme to the known structure of \u201ccousin\u201d enzyme, and then overlaying the location of the new mutations and previously discovered mutations.<\/p>\n
They found that the new mutation was located where the two copies of the protein met \u2014 think the pin that holds a pair of scissors together \u2014 while the well-known mutation was in the functional part of the enzyme \u2014 think the scissors\u2019 blades. By combining one dull blade with a proper pin and one sharp blade without a pin, you can produce a pair of scissors that could work, abet not very well.<\/p>\n
\u201cThe beauty of this work is that we did the benchwork to identify the new mutants and then we did computational modeling to test many, many combinations between these mutants,\u201d Brumos said. \u201cWe could clearly see that if one of the monomers has the mutation on the interaction surface and the other monomer has a mutation in a different domain, then they were able to come together and form a functional enzyme.\u201d<\/p>\n
There are not many examples of this type of complementation in the scientific literature, Stepanova said. Though it likely does occur in nature more frequently than reported.<\/p>\n
An example this type of complementation has been reported in the human disorder phenylketonuria, or PKU. PKU is caused by having mutations in the gene that codes for an enzyme that breaks down the amino acid phenylalanine. Unless on a very special diet, phenylalanine can build up to toxic levels in a baby or individual with PKU, causing a host of neurological issues. However, some individuals with different mutations in each copy of the gene have been reported to show milder symptoms, due to this kind of complementation.<\/p>\n
In addition to highlighting new areas of research, the paper\u2019s findings should serve as a warning for budding geneticists, Stepanova said.<\/p>\n
\u201cThis is a cautionary tale for scientists doing this type of complementation testing,\u201d Stepanova said. \u201cWhen you do this type of test, you\u2019re supposed to use a mutant that cannot make any protein for your control. But sometimes scientists use whatever well-known mutants are available. And if they cross two mutants with mis-sense mutations, there\u2019s a possibility of functional complementation, as we have shown.\u201d<\/p>\n
While some of the other mutants identified in the screen have been published<\/a> by Stepanova and her team, work on others is still ongoing and will be published in the future.<\/p>\nIn addition to Brumos, Stepanova, Alonso and Bobay, Cierra Clark, an undergraduate student who graduated in May 2019, was involved in the study and was an author on the paper.<\/p>\n
The National Science Foundation funded the research.<\/p>\n
Our research addresses grand challenges \u2014 and overcomes them.<\/strong><\/p>\n","protected":false,"raw":"Two wrongs don\u2019t make a right, but sometimes two \u201cbroken\u201d genetic mutations can \u201cfix\u201d each other.\r\n\r\nThat\u2019s what a team of NC State researchers discovered when studying an enzyme involved in making a critical plant growth hormone. Enzymes are proteins made by living organisms to speed up chemical reactions.\r\n\r\nThe enzyme is called ROOTY, because the seedlings have characteristic stumpy and branched roots, said Javier Brumos, a researcher in NC State\u2019s Department of Plant and Microbial Biology.\r\n\r\nBrumos and his colleagues shared their discovery in a recent paper<\/a> published in the journal Plant Physiology<\/em>. They found a new mutation in ROOTY<\/em> that \u201cfixed\u201d a well-studied mutation in the same gene. Then they constructed a computational model to see how the two mutations could fix or complement each other, producing a functional enzyme.\r\n\r\n\u201cFrom the scientific point of view, you\u2019re always looking for novelty, to bring something new to the table,\u201d said Brumos, the first author on the paper. \u201cOur goal was to find new genes that are involved in the production of auxin, a plant hormone. When we identified that the mutation was actually in a well-known gene, we got super depressed. Then we got curious, the mutation is in the same gene: How did it complement the known mutant?\u201d\r\nFirst Up, Then Down<\/strong><\/h3>\r\nBrumos and senior author Anna Stepanova<\/a>, an associate professor of plant and microbial biology, started with Arabidopsis thaliana<\/em>, the widely-used (and fast-growing) plant species favored by plant biologists. They generated a massive collection of seeds with random mutations and then looked at 10-day-old seedlings for specific traits. Specifically, seedlings with curly cotyledons, the first two \u201cleaves\u201d of a plant shoot, or short, stumpy roots.\r\n\r\nThey kept the seedlings with these traits, transferred them to soil, grew them up and bred with themselves. Next, they crossed these \u201cpure bred\u201d mutants with pure bred plants with known mutations in well-studied genes involved in making the plant growth hormone auxin.\r\n\r\n[caption id=\"attachment_178617\" align=\"alignleft\" width=\"460\" class=\"layout_image\"] When a plant with an unknown mutation (TG8B) was crossbred with a plant with a known mutation (RTY1) in an enzyme involved in making a plant growth hormone, the offspring looked normal, suggesting the new mutant complemented the known mutant.[\/caption]\r\n\r\n\u201cAs expected, when we crossed them, most of them looked like the parents, so we knew the new mutation was in the same gene as the known mutation,\u201d Brumos said. \u201cWe could discard those mutants because the gene was already known.\u201d\r\n\r\nHowever, there were some normal-looking seedlings from the crosses, which indicated that the new mutant complemented the known mutant.\r\n\r\n\u201cGenetics 101 suggested that the mutations were in two different genes,\u201d Brumos said. \u201cWe got super excited at that point. We thought we\u2019d found a new gene that was involved in auxin synthesis.\u201d\r\n\r\nHowever, when they mapped and sequenced the new mutation, they were disappointed to learn that the mutation was actually in the well-studied gene, ROOTY<\/em>.\r\n\r\nAfter they recovered from their disappointment, the team began to get curious as to how two mutations in one enzyme could complement each other, as the findings were rather unexpected.\r\n\r\nMany enzymes, including ROOTY, the protein produced by the ROOTY<\/em> gene, work by pairing up with another copy of the protein to form a functional unit \u2014 like two halves of a pair of scissors. The team, with collaborator Douglas Grubb, a group leader at the Leibniz Institute for Plant Biochemistry, had a hypothesis that when a copy of the enzyme with the new mutation paired up with a copy of the enzyme with the well-known mutation, the mis-matched mutant pair was functional. Jose Alonso<\/a>, William Neal Reynolds professor of plant and microbial biology and a long-term collaborator of Stepanova, proposed that the new mutation could disable a different section, or domain, of the enzyme than the well-known mutation.\r\n\r\nTo test this hypothesis, the researchers teamed up with Ben Bobay, a senior research associate at Duke University, to make a computational 3D model of the ROOTY enzyme \u2014 by comparing the normal enzyme to the known structure of \u201ccousin\u201d enzyme, and then overlaying the location of the new mutations and previously discovered mutations.\r\n\r\nThey found that the new mutation was located where the two copies of the protein met \u2014 think the pin that holds a pair of scissors together \u2014 while the well-known mutation was in the functional part of the enzyme \u2014 think the scissors\u2019 blades. By combining one dull blade with a proper pin and one sharp blade without a pin, you can produce a pair of scissors that could work, abet not very well.\r\n\r\n\u201cThe beauty of this work is that we did the benchwork to identify the new mutants and then we did computational modeling to test many, many combinations between these mutants,\u201d Brumos said. \u201cWe could clearly see that if one of the monomers has the mutation on the interaction surface and the other monomer has a mutation in a different domain, then they were able to come together and form a functional enzyme.\u201d\r\n\r\nThere are not many examples of this type of complementation in the scientific literature, Stepanova said. Though it likely does occur in nature more frequently than reported.\r\n\r\nAn example this type of complementation has been reported in the human disorder phenylketonuria, or PKU. PKU is caused by having mutations in the gene that codes for an enzyme that breaks down the amino acid phenylalanine. Unless on a very special diet, phenylalanine can build up to toxic levels in a baby or individual with PKU, causing a host of neurological issues. However, some individuals with different mutations in each copy of the gene have been reported to show milder symptoms, due to this kind of complementation.\r\n\r\nIn addition to highlighting new areas of research, the paper\u2019s findings should serve as a warning for budding geneticists, Stepanova said.\r\n\r\n\u201cThis is a cautionary tale for scientists doing this type of complementation testing,\u201d Stepanova said. \u201cWhen you do this type of test, you\u2019re supposed to use a mutant that cannot make any protein for your control. But sometimes scientists use whatever well-known mutants are available. And if they cross two mutants with mis-sense mutations, there\u2019s a possibility of functional complementation, as we have shown.\u201d\r\n\r\nWhile some of the other mutants identified in the screen have been published<\/a> by Stepanova and her team, work on others is still ongoing and will be published in the future.\r\n\r\nIn addition to Brumos, Stepanova, Alonso and Bobay, Cierra Clark, an undergraduate student who graduated in May 2019, was involved in the study and was an author on the paper.\r\n\r\nThe National Science Foundation funded the research.\r\n\r\nOur research addresses grand challenges \u2014 and overcomes them.<\/strong>"},"excerpt":{"rendered":"NC State researchers discover a new genetic mutation that could \u201cfix\u201d another mutation in the same gene, an enzyme involved in making a plant growth hormone \u2014 after a rollercoaster of ups and downs.<\/p>\n","protected":false},"author":2360,"featured_media":178616,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"source":"","ncst_custom_author":"","ncst_show_custom_author":false,"ncst_dynamicHeaderBlockName":"ncst\/default-post-header","ncst_dynamicHeaderData":"{\"showAuthor\":true,\"showDate\":true,\"showFeaturedVideo\":false,\"caption\":\"Seedlings with mutations in genes involved in making a plant growth hormone have curly cotyledons, the first two \u201cleaves\u201d of a plant shoot, or short roots. \",\"displayCategoryID\":1792}","ncst_content_audit_freq":"","ncst_content_audit_date":"","footnotes":"","_links_to":"","_links_to_target":""},"categories":[1792,1171,1633,1181,1163],"tags":[1863,338],"_ncst_magazine_issue":[],"coauthors":[1677],"class_list":["post-178615","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-cals-weekly","category-faculty-and-staff","category-nc-psi","category-newswire","category-research","tag-_from-newswire-collection-80","tag-department-of-plant-and-microbial-biology"],"displayCategory":{"term_id":1792,"name":"CALS Weekly","slug":"cals-weekly","term_group":0,"term_taxonomy_id":1792,"taxonomy":"category","description":"","parent":0,"count":825,"filter":"raw"},"acf":[],"_links":{"self":[{"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/posts\/178615"}],"collection":[{"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/users\/2360"}],"replies":[{"embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/comments?post=178615"}],"version-history":[{"count":6,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/posts\/178615\/revisions"}],"predecessor-version":[{"id":216695,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/posts\/178615\/revisions\/216695"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/media\/178616"}],"wp:attachment":[{"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/media?parent=178615"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/categories?post=178615"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/tags?post=178615"},{"taxonomy":"_ncst_magazine_issue","embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/_ncst_magazine_issue?post=178615"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/coauthors?post=178615"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}
They kept the seedlings with these traits, transferred them to soil, grew them up and bred with themselves. Next, they crossed these \u201cpure bred\u201d mutants with pure bred plants with known mutations in well-studied genes involved in making the plant growth hormone auxin.<\/p>\n \u201cAs expected, when we crossed them, most of them looked like the parents, so we knew the new mutation was in the same gene as the known mutation,\u201d Brumos said. \u201cWe could discard those mutants because the gene was already known.\u201d<\/p>\n However, there were some normal-looking seedlings from the crosses, which indicated that the new mutant complemented the known mutant.<\/p>\n \u201cGenetics 101 suggested that the mutations were in two different genes,\u201d Brumos said. \u201cWe got super excited at that point. We thought we\u2019d found a new gene that was involved in auxin synthesis.\u201d<\/p>\n However, when they mapped and sequenced the new mutation, they were disappointed to learn that the mutation was actually in the well-studied gene, ROOTY<\/em>.<\/p>\n After they recovered from their disappointment, the team began to get curious as to how two mutations in one enzyme could complement each other, as the findings were rather unexpected.<\/p>\n Many enzymes, including ROOTY, the protein produced by the ROOTY<\/em> gene, work by pairing up with another copy of the protein to form a functional unit \u2014 like two halves of a pair of scissors. The team, with collaborator Douglas Grubb, a group leader at the Leibniz Institute for Plant Biochemistry, had a hypothesis that when a copy of the enzyme with the new mutation paired up with a copy of the enzyme with the well-known mutation, the mis-matched mutant pair was functional. Jose Alonso<\/a>, William Neal Reynolds professor of plant and microbial biology and a long-term collaborator of Stepanova, proposed that the new mutation could disable a different section, or domain, of the enzyme than the well-known mutation.<\/p>\n To test this hypothesis, the researchers teamed up with Ben Bobay, a senior research associate at Duke University, to make a computational 3D model of the ROOTY enzyme \u2014 by comparing the normal enzyme to the known structure of \u201ccousin\u201d enzyme, and then overlaying the location of the new mutations and previously discovered mutations.<\/p>\n They found that the new mutation was located where the two copies of the protein met \u2014 think the pin that holds a pair of scissors together \u2014 while the well-known mutation was in the functional part of the enzyme \u2014 think the scissors\u2019 blades. By combining one dull blade with a proper pin and one sharp blade without a pin, you can produce a pair of scissors that could work, abet not very well.<\/p>\n \u201cThe beauty of this work is that we did the benchwork to identify the new mutants and then we did computational modeling to test many, many combinations between these mutants,\u201d Brumos said. \u201cWe could clearly see that if one of the monomers has the mutation on the interaction surface and the other monomer has a mutation in a different domain, then they were able to come together and form a functional enzyme.\u201d<\/p>\n There are not many examples of this type of complementation in the scientific literature, Stepanova said. Though it likely does occur in nature more frequently than reported.<\/p>\n An example this type of complementation has been reported in the human disorder phenylketonuria, or PKU. PKU is caused by having mutations in the gene that codes for an enzyme that breaks down the amino acid phenylalanine. Unless on a very special diet, phenylalanine can build up to toxic levels in a baby or individual with PKU, causing a host of neurological issues. However, some individuals with different mutations in each copy of the gene have been reported to show milder symptoms, due to this kind of complementation.<\/p>\n In addition to highlighting new areas of research, the paper\u2019s findings should serve as a warning for budding geneticists, Stepanova said.<\/p>\n \u201cThis is a cautionary tale for scientists doing this type of complementation testing,\u201d Stepanova said. \u201cWhen you do this type of test, you\u2019re supposed to use a mutant that cannot make any protein for your control. But sometimes scientists use whatever well-known mutants are available. And if they cross two mutants with mis-sense mutations, there\u2019s a possibility of functional complementation, as we have shown.\u201d<\/p>\n While some of the other mutants identified in the screen have been published<\/a> by Stepanova and her team, work on others is still ongoing and will be published in the future.<\/p>\n In addition to Brumos, Stepanova, Alonso and Bobay, Cierra Clark, an undergraduate student who graduated in May 2019, was involved in the study and was an author on the paper.<\/p>\n The National Science Foundation funded the research.<\/p>\n Our research addresses grand challenges \u2014 and overcomes them.<\/strong><\/p>\n","protected":false,"raw":"Two wrongs don\u2019t make a right, but sometimes two \u201cbroken\u201d genetic mutations can \u201cfix\u201d each other.\r\n\r\nThat\u2019s what a team of NC State researchers discovered when studying an enzyme involved in making a critical plant growth hormone. Enzymes are proteins made by living organisms to speed up chemical reactions.\r\n\r\nThe enzyme is called ROOTY, because the seedlings have characteristic stumpy and branched roots, said Javier Brumos, a researcher in NC State\u2019s Department of Plant and Microbial Biology.\r\n\r\nBrumos and his colleagues shared their discovery in a recent paper<\/a> published in the journal Plant Physiology<\/em>. They found a new mutation in ROOTY<\/em> that \u201cfixed\u201d a well-studied mutation in the same gene. Then they constructed a computational model to see how the two mutations could fix or complement each other, producing a functional enzyme.\r\n\r\n\u201cFrom the scientific point of view, you\u2019re always looking for novelty, to bring something new to the table,\u201d said Brumos, the first author on the paper. \u201cOur goal was to find new genes that are involved in the production of auxin, a plant hormone. When we identified that the mutation was actually in a well-known gene, we got super depressed. Then we got curious, the mutation is in the same gene: How did it complement the known mutant?\u201d\r\n NC State researchers discover a new genetic mutation that could \u201cfix\u201d another mutation in the same gene, an enzyme involved in making a plant growth hormone \u2014 after a rollercoaster of ups and downs.<\/p>\n","protected":false},"author":2360,"featured_media":178616,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"source":"","ncst_custom_author":"","ncst_show_custom_author":false,"ncst_dynamicHeaderBlockName":"ncst\/default-post-header","ncst_dynamicHeaderData":"{\"showAuthor\":true,\"showDate\":true,\"showFeaturedVideo\":false,\"caption\":\"Seedlings with mutations in genes involved in making a plant growth hormone have curly cotyledons, the first two \u201cleaves\u201d of a plant shoot, or short roots. \",\"displayCategoryID\":1792}","ncst_content_audit_freq":"","ncst_content_audit_date":"","footnotes":"","_links_to":"","_links_to_target":""},"categories":[1792,1171,1633,1181,1163],"tags":[1863,338],"_ncst_magazine_issue":[],"coauthors":[1677],"class_list":["post-178615","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-cals-weekly","category-faculty-and-staff","category-nc-psi","category-newswire","category-research","tag-_from-newswire-collection-80","tag-department-of-plant-and-microbial-biology"],"displayCategory":{"term_id":1792,"name":"CALS Weekly","slug":"cals-weekly","term_group":0,"term_taxonomy_id":1792,"taxonomy":"category","description":"","parent":0,"count":825,"filter":"raw"},"acf":[],"_links":{"self":[{"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/posts\/178615"}],"collection":[{"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/users\/2360"}],"replies":[{"embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/comments?post=178615"}],"version-history":[{"count":6,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/posts\/178615\/revisions"}],"predecessor-version":[{"id":216695,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/posts\/178615\/revisions\/216695"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/media\/178616"}],"wp:attachment":[{"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/media?parent=178615"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/categories?post=178615"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/tags?post=178615"},{"taxonomy":"_ncst_magazine_issue","embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/_ncst_magazine_issue?post=178615"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/cals.ncsu.edu\/wp-json\/wp\/v2\/coauthors?post=178615"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}First Up, Then Down<\/strong><\/h3>\r\nBrumos and senior author Anna Stepanova<\/a>, an associate professor of plant and microbial biology, started with Arabidopsis thaliana<\/em>, the widely-used (and fast-growing) plant species favored by plant biologists. They generated a massive collection of seeds with random mutations and then looked at 10-day-old seedlings for specific traits. Specifically, seedlings with curly cotyledons, the first two \u201cleaves\u201d of a plant shoot, or short, stumpy roots.\r\n\r\nThey kept the seedlings with these traits, transferred them to soil, grew them up and bred with themselves. Next, they crossed these \u201cpure bred\u201d mutants with pure bred plants with known mutations in well-studied genes involved in making the plant growth hormone auxin.\r\n\r\n[caption id=\"attachment_178617\" align=\"alignleft\" width=\"460\" class=\"layout_image\"]
When a plant with an unknown mutation (TG8B) was crossbred with a plant with a known mutation (RTY1) in an enzyme involved in making a plant growth hormone, the offspring looked normal, suggesting the new mutant complemented the known mutant.[\/caption]\r\n\r\n\u201cAs expected, when we crossed them, most of them looked like the parents, so we knew the new mutation was in the same gene as the known mutation,\u201d Brumos said. \u201cWe could discard those mutants because the gene was already known.\u201d\r\n\r\nHowever, there were some normal-looking seedlings from the crosses, which indicated that the new mutant complemented the known mutant.\r\n\r\n\u201cGenetics 101 suggested that the mutations were in two different genes,\u201d Brumos said. \u201cWe got super excited at that point. We thought we\u2019d found a new gene that was involved in auxin synthesis.\u201d\r\n\r\nHowever, when they mapped and sequenced the new mutation, they were disappointed to learn that the mutation was actually in the well-studied gene, ROOTY<\/em>.\r\n\r\nAfter they recovered from their disappointment, the team began to get curious as to how two mutations in one enzyme could complement each other, as the findings were rather unexpected.\r\n\r\nMany enzymes, including ROOTY, the protein produced by the ROOTY<\/em> gene, work by pairing up with another copy of the protein to form a functional unit \u2014 like two halves of a pair of scissors. The team, with collaborator Douglas Grubb, a group leader at the Leibniz Institute for Plant Biochemistry, had a hypothesis that when a copy of the enzyme with the new mutation paired up with a copy of the enzyme with the well-known mutation, the mis-matched mutant pair was functional. Jose Alonso<\/a>, William Neal Reynolds professor of plant and microbial biology and a long-term collaborator of Stepanova, proposed that the new mutation could disable a different section, or domain, of the enzyme than the well-known mutation.\r\n\r\nTo test this hypothesis, the researchers teamed up with Ben Bobay, a senior research associate at Duke University, to make a computational 3D model of the ROOTY enzyme \u2014 by comparing the normal enzyme to the known structure of \u201ccousin\u201d enzyme, and then overlaying the location of the new mutations and previously discovered mutations.\r\n\r\nThey found that the new mutation was located where the two copies of the protein met \u2014 think the pin that holds a pair of scissors together \u2014 while the well-known mutation was in the functional part of the enzyme \u2014 think the scissors\u2019 blades. By combining one dull blade with a proper pin and one sharp blade without a pin, you can produce a pair of scissors that could work, abet not very well.\r\n\r\n\u201cThe beauty of this work is that we did the benchwork to identify the new mutants and then we did computational modeling to test many, many combinations between these mutants,\u201d Brumos said. \u201cWe could clearly see that if one of the monomers has the mutation on the interaction surface and the other monomer has a mutation in a different domain, then they were able to come together and form a functional enzyme.\u201d\r\n\r\nThere are not many examples of this type of complementation in the scientific literature, Stepanova said. Though it likely does occur in nature more frequently than reported.\r\n\r\nAn example this type of complementation has been reported in the human disorder phenylketonuria, or PKU. PKU is caused by having mutations in the gene that codes for an enzyme that breaks down the amino acid phenylalanine. Unless on a very special diet, phenylalanine can build up to toxic levels in a baby or individual with PKU, causing a host of neurological issues. However, some individuals with different mutations in each copy of the gene have been reported to show milder symptoms, due to this kind of complementation.\r\n\r\nIn addition to highlighting new areas of research, the paper\u2019s findings should serve as a warning for budding geneticists, Stepanova said.\r\n\r\n\u201cThis is a cautionary tale for scientists doing this type of complementation testing,\u201d Stepanova said. \u201cWhen you do this type of test, you\u2019re supposed to use a mutant that cannot make any protein for your control. But sometimes scientists use whatever well-known mutants are available. And if they cross two mutants with mis-sense mutations, there\u2019s a possibility of functional complementation, as we have shown.\u201d\r\n\r\nWhile some of the other mutants identified in the screen have been published<\/a> by Stepanova and her team, work on others is still ongoing and will be published in the future.\r\n\r\nIn addition to Brumos, Stepanova, Alonso and Bobay, Cierra Clark, an undergraduate student who graduated in May 2019, was involved in the study and was an author on the paper.\r\n\r\nThe National Science Foundation funded the research.\r\n\r\nOur research addresses grand challenges \u2014 and overcomes them.<\/strong>"},"excerpt":{"rendered":"