Characterization of caffeoyl coenzyme A O-methyltransferase genes in maize AND The role of maysin in maize as a defense response to herbivory

Open Access
Author:
Marchinchin, Joseph Robert
Graduate Program:
Agronomy
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
November 09, 2011
Committee Members:
  • Dawn S Luthe, Thesis Advisor
Keywords:
  • herbivory
  • maysin
  • predicted protein structure
  • allelic differences
  • S:G ratio
  • lignin modification
  • CCoAOMT
  • antibiosis
  • bioassays
  • corn earworm
  • fall armyworm
  • maize
Abstract:
PART A: Characterization of caffeoyl coenzyme A O-methyltransferase (CCoAOMT) genes in maize. CCoAOMT is an important enzyme in the lignin biosynthetic pathway that catalyzes the conversion of caffeoyl-CoA into feruloyl-CoA. CCoAOMT activity has been linked to the production of guaiacyl lignin, one of the three types of monolignol subunits present in the cell wall. Aspergillus flavus is a major pathogen of maize due to its production of aflatoxin B1, a potent mycotoxin linked to cancer incidence and developmental disorders in animals. Previous studies involving two maize lines known for their resistance and susceptibility to A. flavus pathogenicity (Mp313E and Sc212M, respectfully) have shown CCoAOMT to be 66 times more abundant in the developing rachis tissue of Mp313E (r) than Sc212M (s). Numerous studies suggest CCoAOMT is capable of altering both lignin composition and overall quantity within the cell wall, leading to the hypothesis that such differences affect cell wall digestibility and confer inherent resistance against A. flavus in Mp313E. As of October 2011, 5 unique CCoAOMT genes are believed to exist within the maize genome, however only CCoAOMT 1 has been characterized. Using template cDNA sequences from CCoAOMTs 2, 3, 4 and 5 retrieved on NCBI, CCoAOMTs 2, 3, 4 and 5 in Mp313E and Sc212M were amplified, cloned and sequenced from both gDNA and cDNA. The final protein sequence for each CCoAOMT gene was deduced. Nucleotide and amino acid alignments revealed few allelic differences across R and S genotypes. An alignment among all four CCoAOMTs shows moderate amino acid variation while simultaneously demonstrating a high level of conservatism across each gene. The most significant variation across CCoAOMT peptides occurs near the N-terminus. The molecular weight and isoelectric point of each deduced protein was determined. Further studies testing gene expression and enzyme activity are recommended to better characterize these four CCoAOMT genes and the subsequent enzymes they encode. PART B: The role of maysin in maize as a defense response to herbivory. Lepidopteran pests are directly responsible for significant seed losses in maize annually. Maysin is a C-glycosyl flavone produced primarily in silk tissue in a variety of maize lines. First isolated and characterized in 1979, the accumulation of maysin in fresh silks has been reported to confer resistance against corn earworm. Maysin’s presumed conversion into “maysin-o-quinone” in the presence of polyphenol oxidase results in its binding to key nucleophilic amino acids that subsequently become “biounavailable” to the developing caterpillar, hence the caterpillar starves. While numerous studies suggest that maysin is capable of hindering caterpillar growth at low concentrations (less than 1% by fresh weight composition), others refute this claim, presenting conflicting findings. Using corn earworm (H. zea) and fall armyworm (S. frugiperda) as model organisms, artificial caterpillar diets supplemented with purified maysin at various concentrations were used to assess caterpillar growth in bioassays. Potential interactive effects between maysin concentration, protein concentration, protein source (soy vs. casein) and caterpillar species were also examined. Overall, maysin was found to significantly reduce caterpillar growth in both species at most tested concentrations (ranging from 0.1% to 0.5%). No correlation between maysin concentration and mortality was established. Interactive effects among any combinations of variables were not detected in any bioassay. Corn earworm appeared better adapted to tolerate dietary maysin than fall armyworm, growing to about the same mass in the presence of 0.25% maysin as fall armyworm did in the presence of 0.1% maysin. Collectively, this study suggests that maysin is capable of significantly hindering caterpillar growth, even at concentrations as little as 0.1% per fresh weight tissue.