Wednesday, May 2, 2012

How Glyphosate and Glyphosate Resistant Plants Work

Spring is finally here, with SUN!, warm weather, green grass and the flowers.  But with all of this spring time greatness comes every gardener’s and farmer’s enemy; weeds.  Weeds come in all shapes and sizes, from short and bushy to tall and thin, they are sore on the eyes of anyone who is trying to grow something.  Weeds compete for nutrients, sun, and water with the other plants around them.  They have developed by natural selection for weeds that grow taller and faster than the desired plants by taking up more of the nutrients and water.  How does one get rid of these monsters?  If you have a smaller garden or flower bed the easiest way to kill all of the weeds is to pull them out manually, or with a hoe.  But you can’t do that to an 80 acre field of corn or soybeans unless you want to spend the next few days doing it.  To solve this problem agricultural companies have made and developed herbicide chemicals that kill the targeted weeds.  One of the most known and used herbicide is Round-up.  It’s active ingredient or the part of it that actually does the work is called Glyphosate.  Glyphosate is a chemical compound that was developed and sold by Monsanto under the name of Roundup, and has been and is the most used herbicide in the United States.  So what is the molecular basis for the Herbicide RoundUp?  And if this is used to kill plants then how are crops resistant to it?
Shikimate Pathway

               Glyphosate’s mode of action or how it works is that it inhibits or stops the  production of an enzyme that is involved in the synthesis of aromatic amino acids.  This happens in the Shikimate Pathway.  Amino acids serve as building blocks of proteins that are used throughout the entire plant.   Of the 20 amino acids only three are inhibited; tyrosine, tryptophan, and phenylalanine because they are aromatic and are made through the Shikimate Pathway.  In plants Tyrosine is mainly used in Photosynthesis.  It is actually in chloroplasts where photosystem II is in the plant cells.  Tyrosine acts as an electron donor in the making of NADPH in photosynthesis.  It is made in the shikimate pathway.  Tryptophan is another amnio acid that is inhibited and it is used as building blocks for proteins.  Phenylalanine is the starting compound of flavonoid biosynthesis.  It can also be converted through chemical pathways to make cinnamic acid.  The flavonoids are the most important plant pigments for flower color, especially yellow or red/blue petals.  Tyrosine, Tryptophan, and Phenylalanie are made through a complicated mechanism that starts with chorismate (Wikipedia).

(Below shows EPSP synthase working normally in Marvin Sketch)

                Photosynthesis is the process that plants do to create sugars to use for metabolism and growth.  Besides making sugars, the other end product is oxygen that is used by mammals to breath.  Photosynthesis takes place in the chloroplasts which are organelles that are found only in plant cells or some eukaryotic organisms.  In the chloroplast there are chlorophyll cells that absorb the sun’s energy using light’s wavelengths.  From this light energy, ATP and NADPH are made which are used in respiration that produces the byproduct oxygen.  Photosystem II and Photosystem I happen in the light reaction, where the light energy is absorbed and electrons are transported from Photosystem II to Photosystem I which then makes the NADPH.  
Light Reaction in Photosynthesis
Shikimate pathway uses the metabolism of the carbohydrates to make aromatic compounds by biosynthesis.  There are seven metabolic steps,  phosphoenolpyruvate and erythrose 4 phosphate are converted to chorismate, the precursor of the aromatic amino acids and many aromatic secondary metabolites.  All of the pathway’s intermediates are used to make aromatic amino acids and are substrates for other metabolic pathways.  The Shikimate pathway is only found in plants and microorganisms, never in humans.  Glyphosate disrupts this process between Shikimate and the formation of Chorismate.  Glyphosate blocks the enzyme 5-enolpyruvylshikimate-3-phosphate synthase, also known as EPSP synthase.  EPSP synthase takes a phosphoric acid from the combining Shikimate and phosphoenolpyruvate (PEP) to make EPSE that goes on in the mechanism to work with chorismate synthase which gets rid of another phosphoric acid to make chorismate, the precursor to many amino acids (Herrmann, and Weaver).

The Shikimate pathway is usually found in the actively growing part of the plant.  Glyphosate is absorbed through the foliage or leaves of plants and transported through the cells to the growing points, because of this glyphosate is only effective on growing plants.  You may be wondering since this inhibits amino acid synthesis, how the desired crop survives while the weeds do not.  In regular plants the EPSP synthase catalyzes the transfer of PEP to the hydroxyl group at the five carbon position.

                                             (The figure above is the Jmole for the EPSP synthase) 
 In the 1990s there was a stand of E. coli that was found in a waste area at a glyphosate production facility, that was glyphosate resistant, called Agrobacterium sp strain CP4.  From this the company was able to form a synthetic molecule that included the amino acids Ala-100-Gly CP4.

(The Jmol below shows the ESPS synthase that is the mutant glyphosate resistant system)
In the study done by Funke about the molecular make up of herbicide resistant Round up ready crops shows that the effect of potassium ions on the enzyme’s activity appear to be selective toward PEP utilization, showing that CP4 EPSP synthase is the prototypic class II EPSP synthase, because the catalytic efficiency of the reaction remains basicially unaltered in the present of glyphosate.  This allows the CP4 EPSP synthase to work to allow plants to be herbicide resistant (Funke, Han, and et al).  This new EPSPS gene encode for a polypeptide that contains a chloroplast transit peptide which enables the EPSPS polypeptide to be transported into a chloroplast inside of a plant cell.  Once it is in the chloroplast it has the ability to work properly allowing the plant to make photosynthesis (Shah). So if we have found a way for our crops to be resistance is there a way that plants could become resistant to glyphosate?  

The answer is yes, there are resistant weeds.  There are weeds species that have a lot of genetic diversity in their genome, and example of this is Amaranthus tuberculatus, or Tall Waterhemp.  Tall Waterhemp is a dioecious plant, that produces on average one million seeds per plant, and is a species that has separate male and female plants, because of this they are not self-fertilized.  Because these plants have to cross pollinate to be fertilized, there is a large flow of genes from plant to plant.  Some of these genes carry the alleles that are needed to override the ESPS inhibition that glyphosate creates.  Spraying with Round up frequently causes selection for glyphosate resistant plants, because all of the non-resistant plants are dead.  Usually the resistant gene is the recessive gene, to make it easy to explain we need to remember high school genetics, specifically Medelian genetics.  To be homozygous recessive you need both of the recessive alleles, in this example are the glyphosate resistant alleles.  Non-resistant genes have the dominate allele so the non-resistant trait is expressed.  With selecting for the resistant gene, (not on purpose!) you are allowing the homozygous recessive plants to mate with each other and create millions of other glyphosate resistant weeds.  A study done by the Tom Gains group out of Colorado State University looked at one way that weeds could naturally be resistant to glyphosate.  They discovered that with glyphosate selection pressure that they were finding more and more Amaranthus palmeri plants to have an over expression of the EPSP synthase gene.  They used quantitative PCR to measure the relative copy numbers of the EPSP synthase gene and found that resistant plants contained five times to 160 times more copies of the EPSP synthase gene than their susceptible counterparts.  They also found that this mutation was heritable and that it could be easily passed down to later generations (Gaines, Zhang, and et at).  A way to help slow this process is to use different herbicides with different modes of action or different ways to kill the plant.  Other ways include using cultural practices that include crop rotation, planting a different crop the next year, for example, corn this year, beans next year and corn the year after that, and using tillage equipment to keep the row clean while the crop is still short.  Resistant weeds are a growing problem in agriculture, but farmers and agriculturalist are combating this problem with changing the mode of action and cultural practices that were mentioned above.           

Glyphosate had been a great advancement in the control of weeds for farmers and gardeners.  With it being a broad spectrum weed controller it allows farmers to only have to spray twice a growing season instead of spraying every three weeks, like it was before Round up.  RoundUp Ready plants, plants that are resistant to glyphosate, have been a great advancement for farmers as well.  Farmers can plant these crops and not have to worry about the crops being damaged from the glyphosate that kills the weeds.  Even with the problems that are created by glyphosate resistant weeds, glyphosate has been a great product that has made farmer’s lives easier and has allowed for advancement in agriculture.

Works Cited 

Funke, Todd, Huijong Han, et al. "Molecular Basis for the herbicide resistance of Round up Ready crops ." Proceddings of the National Academy of Sciences of the United States of America. 103.35 (2006): 13010-13015. Web.  1 April 2012.

Gaines, Todd, Wenli Zhang, et al. "Gene amplification confers glyphosate resistance in Amaranthus palmeri." Proceddings of the National Academy of Sciences of the United States of America. 107.3 (19, January 2010): 1029-1034. Web. 30, April 2012.

Herrmann, KM, and LM Weaver. "The Shikimate Pathway." Annual Review Plant Physiologyand Plant Molecular Biology . 50.June (1999): 473-503. Web. 12 March, 2012.
Shah, Dilip, Stephen Rogers, et al. United States of America. United States Patent. Glyphosate-resistant plants. 10, July 1990. Web.
"Glyphosate." Wikipedia. Wikipedia, 12 March 2012. Web. 13 March 2012. <>.

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