Study: Sorghum wards off pests by releasing hydrogen cyanide

December 18, 2013

Brian Dilkes, at left, and Mitch Tuinstra with sorghum plants. (Purdue Agricultural Communication photo/Tom Campbell)

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WEST LAFAYETTE, Ind. - Purdue University researchers have confirmed the long-held hypothesis that sorghum deters insects from feeding on its leaves by releasing hydrogen cyanide.

Mitch Tuinstra and Brian Dilkes found that insects preferred the leaves of a mutant sorghum plant with an abnormally slow release of cyanide to those of a wild-type sorghum plant with a normal cyanide-release rate. Fall army worms fed on the leaves of the mutant sorghum even though the leaves contained similar levels of dhurrin - the chemical compound responsible for synthesizing hydrogen cyanide - as those in normal sorghum plants.

"This study separates for the first time the accumulation of dhurrin from the release of hydrogen cyanide," said Dilkes, assistant professor of horticulture and landscape architecture. "Both the mutant and normal sorghum plants contain dhurrin, but it's the rate of cyanide release that causes the insects to avoid one in favor of the other. It's a beautiful interaction between animal behavior and plant chemistry."

Sorghum bicolor, the cultivated species of sorghum, is a critically important cereal grass used for food and animal forage in many parts of the developing world and is a promising bioenergy crop in the U.S. Its ability to thrive in arid environments makes it a more water-efficient crop than corn.

While the grain of sorghum is edible, its leaves can sometimes contain levels of hydrogen cyanide that are toxic to humans and animals. Livestock producers have long known that feeding sorghum leaves harvested at certain growth stages, and particularly under stress conditions such as drought, can result in cyanide poisoning of livestock. When properly managed, however, sorghum leaves can be safe forage for cattle.

Identification of genes that control cyanide production and release could lead to the development of cyanide-free sorghum plants.

Tuinstra and Dilkes identified a sorghum mutant with an exceptionally slow cyanide-release rate. They located the gene responsible for the defect by using next-generation sequencing, a technique that randomly generates short sequences from a genome - the total genetic content of an organism - and stitches them back together. Next-generation sequencing works like a text editor, said Tuinstra, professor of plant breeding and genetics.

"It's just like taking a 10-word sentence from a book and asking where it belongs," he said. "It finds the location of a specific sequence inside the species genome. The mutation is like a misplaced period in the middle of a sentence - it signals the reader to stop. In the case of the sorghum mutant, it halts the production of a functional protein."

The sequencing technique allowed Tuinstra and Dilkes to identify the single nucleotide within the sorghum genome of 790 million base pairs that slowed the release of cyanide in the mutant plant.

"This study is an example of how new methods in DNA sequencing can now be used to unlock the genetic mechanisms of sorghum performance," Tuinstra said.

After cloning the mutant, the researchers tested insect feeding preference by releasing fall army worms onto mutant and normal sorghum plants. Though both types of sorghum contained normal levels of dhurrin, the insects avoided the normal sorghum plants, settling and feeding on the leaves of the mutant sorghum instead. While the mutant contains the compounds necessary to generate cyanide, it cannot release cyanide quickly enough to ward off pests, Tuinstra said.

Next-generation sequencing is more often used in plant species with genomes much smaller than sorghum. The study clears the way to use advanced sequencing techniques to identify genes and gene functions in plants with large genomes, Dilkes said.

"We've demonstrated that these sequencing tools are robust enough to apply to organisms with complex genomes," he said. "If we can use them in sorghum, we can use them in other crops. In terms of identifying genes of interest in complex organisms, we're open for business."

The paper was published in Genetics and is available at http://www.genetics.org/content/195/2/309.full.pdf+html?sid=39121ec4-c3f7-43f8-a7d0-8e5107d03b40 .

The research was conducted using funds from the U.S. Department of Energy, the International Sorghum and Millet Collaboration Research Support Program and an Agriculture and Food Research Initiative Competitive Grant.

Writer: Natalie van Hoose, 765-496-2050, nvanhoos@purdue.edu

Sources: Brian Dilkes, 765-494-2584, bdilkes@purdue.edu

Mitch Tuinstra, 765-494-9093, drmitch@purdue.edu

Related website:

Purdue University Agronomy Department

ABSTRACT Forward genetics by genome sequencing reveals that rapid cyanide release deters insect herbivory of Sorghum bicolor Kartikeya Krothapalli 1; Elizabeth M. Buescher 2; Xu Li 3*; Elliot Brown 2; Clint Chapple 3; Brian P. Dilkes 2; Mitchell R. Tuinstra 1 1 Department of Agronomy, Purdue University, West Lafayette, Indiana, United States of America 2 Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America 3 Department of Biochemistry, Purdue University, West Lafayette, Indiana, United States of America *Current address: Plants for Human Health Institute; Department of Plant Biology, North Carolina State University, Kannapolis, North Carolina, United States of America E-mail: drmitch@purdue.edu; bdilkes@purdue.edu Whole genome sequencing has allowed rapid progress in the application of forward genetics in model species. In this study, we demonstrated an application of Next-Generation Sequencing for forward genetics in a complex crop genome. We sequenced an ethyl methanesulfonate-induced mutant of Sorghum bicolor defective in hydrogen cyanide release and identified the causal mutation. A workflow identified the causal polymorphism relative to the reference BTx623 genome by integrating data from single nucleotide polymorphism identification, prior information about candidate gene(s) implicated in cyanogenesis, mutation spectra, and polymorphisms likely to affect phenotypic changes. A point mutation resulting in a premature stop codon in the coding sequence of dhurrinase2, a protein involved in the dhurrin catabolic pathway, was responsible for the acyanogenic phenotype. Cyanogenic glucosides are not cyanogenic compounds but their cyanohydrins derivatives do release cyanide. The mutant accumulated the glucoside, dhurrin, but failed to efficiently release cyanide upon tissue disruption. Thus, we tested the effects of cyanide release on insect herbivory in a genetic background in which accumulation of cyanogenic glucoside is unchanged. Insect preference choice experiments and herbivory measurements demonstrate a deterrent effect of cyanide release capacity, even in the presence of wild-type levels of cyanogenic glucoside accumulation. Our gene cloning method substantiates the value of 1.) a sequenced genome; 2.) a strongly penetrant and easily measurable phenotype; and 3.) a workflow to pinpoint a causal mutation in crop genomes and accelerate in the discovery of gene function in the post genomic era.

Ag Communications: (765) 494-2722;

Keith Robinson, robins89@purdue.edu

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