Characterization of 4-hydroxycinnamic acid: CoA ligase, anthocyanins and pathogen ingress in resistant and susceptible maize cultivars

John D Hipskind, Purdue University

Abstract

Phenylpropanoid metabolism in maize cultivars B73$\sb{Ht}$ (susceptible) and B73$\sb{Ht\ rhm}$ (resistant) is conspicuously affected following inoculation with the fungus Bipolaris maydis race O. The purpose of this research was to examine the role of 4-hydroxycinnamic acid: CoA ligase (4CL) in the disease interaction from infected host tissues and to establish whether isozymes are induced. Anion exchange and hydrophobic interaction chromatography of protein preparations extracted in bulk from maize leaves infected with B. maydis resulted in over a 600-fold purification of 4CL. The native enzyme had an estimated molecular weight of 51.6 kDa. Kinetic analysis of enzyme activities with various phenylpropanoid substrates demonstrated there is little preferential substrate-specificity among phenylpropanoids tested. The enzyme exhibited no activity when assayed with sinapic acid. No evidence for multiple forms was found in the present investigation, suggesting that the maize 4CL is not responsible for metabolic channeling of phenylpropanoids and their distribution within the cell which occurs in response to infection. The second goal of this research was to chemically characterize anthocyanin pigments which often accumulate in uninfected cells adjacent to restricted lesions in the resistant B73$\sb{Ht\ rhm}$ cultivar. Results showed that anthocyanins begin to accumulate by 32 hours post-inoculation and continue to accumulate through 72 hours. Plasma Desorption Mass Spectrometry (PDMS) of anthocyanins that accumulate in the healthy leaf sheath tissues revealed a major ion with a molecular mass of 621. A search of the literature identified this 621 ion as a cyanidin 3-dimalonylglucoside. Importantly, PDMS of anthocyanins isolated from infected tissues showed the identical 621 ion. The final goal of this research was to use fluorescent staining and microscopic examination of B. maydis hyphae at various times after infection to delineate the time when fungal ingress is arrested. Computer analysis revealed that the growth of B. maydis in the resistant cultivar is restricted prior to 18 hours post-inoculation. However, analysis of the susceptible tissues showed an almost linear growth of fungal hyphae. Importantly, this investigation shows that the cessation of pathogen ingress in the resistant cultivar occurs much earlier than the previously reported accumulation of phenylpropanoids.

Degree

Ph.D.

Advisors

Nicholson, Purdue University.

Subject Area

Plant pathology|Botany

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