1.9.13
DETERMINATION OF THE TEMPORAL SYNTHESIS OF SORGHUM PHYTOALEXINS USING PHOTODIODE ARRAY-HPLC AND MALDI-MASS SPECTROMETRY

PS WHARTON1 and RL NICHOLSON1

1Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907-1155, USA

Background and objectives
Previous investigations have demonstrated the rapid accumulation of four phytoalexins in sorghum after inoculation with various fungal pathogens and non-pathogens. These have been identified as the 3-deoxyanthocyanidins apigeninidin, luteolinidin, a caffeic acid ester of arabinosyl-5-O-apigeninidin, and 5-methoxy-luteolinidin [1]. Although these compounds are present in detectable amounts in sorghum by 24 h after inoculation, little is known about their pattern of accumulation between 0 and 24 h. The use of HPLC coupled to a highly sensitive photodiode array detector (HPLC-PDA) enables us to detect phenylpropanoid/ flavonoid compounds absorbing in the range 200-600 nm. This has made it possible for us to document the accumulation of not just the 3-deoxyanthocyanidin phytoalexins, but also other phenols which have been synthesized in response to inoculation.

It has been shown previously that matrix-assisted laser desorption ionization (MALDI) mass spectrometry can be used in the analysis of 3-deoxyanthocyanidins and anthocyanins present in crude extracts from sorghum plant tissue. Furthermore, sensitivities to the level of 15 pmol/ml were easily attained for 3-deoxyanthocyanidins present in crude sorghum extracts [2]. Through its capacity for the analysis of such small quantities of these compounds in unpurified samples, MALDI provides a sensitive means for the detection of these and other flavonoid pigments.

Results and conclusions
HPLC of crude extracts from etiolated sorghum mesocotyls revealed that apigeninidin was the first 3-deoxyanthocyanidin to be synthesized, being present in detectable amounts by 8 h after inoculation with the non-pathogenic fungus Cochliobolus heterostrophus. This was followed by detectable amounts of the caffeic acid ester of apigeninidin at 10 h and luteolinidin at 12 h post inoculation (h.p.i). The levels of the phytoalexins rose steadily, with apigeninidin being the most abundant between 10 and 18 h.p.i. However, the rate of accumulation of luteolinidin started to increase after 14 h and by 20 h the amount of luteolinidin was approximately equal to that of apigeninidin. By 24 h.p.i. luteolinidin was the most abundant phytoalexin, followed by apigeninidin and the caffeic acid ester of apigeninidin with 5-methoxy-luteolinidin present in barely detectable amounts. A fifth unknown compound was also detected at 24 h by HPLC. MALDI analysis indicated that this compound was 7-methoxy-apigeninidin. MALDI analysis also confirmed the identities of each of the other phytoalexins.

We have used the two techniques above to fully document the temporal synthesis of the sorghum 3-deoxyanthocyanidins, and hope in the future to use the combination of these two techniques to identify and chemically characterize the intermediates involved in 3-deoxyanthocyanidin synthesis.

References
1. Weiergang I, Hipskind JD, Nicholson RL, 1996. Physiological and Molecular Plant Pathology 49, 377-388.
2. Sugui JA, Bonham C, Lo SC et al., 1998. Phytochemistry (in press).