A 13C-Pulse-labeling study of phytoalexin biosynthesis in hypersensitively responding cotton cotyledons

Abstract

Time courses of multiplication of an incompatible strain of Xanthomonas campestris pv. malvacearum, accumulation of phytoalexins, and development of phytoalexin-rich, yellow-green fluorescent, hypersensitively necrotic cells were followed in cotton cotyledons. Occasionally, hypersensitively responding palisade cells were seen to pass through a stage in which the yellow-green fluorescence, due to the phytoalexins lacinilene C and its methyl ether, was confined to the cytoplasmic layer surrounding an intact vacuole, but more often, when cells became yellow-green fluorescent, the fluorescence appeared throughout the cell. The period of most rapid increase in fluorescent cell numbers, 48-72 h post-inoculation, was also the period of most rapid increase in phytoalexin content, and the rate of bacterial multiplication began to slow down at this time. Stable 13C isotope incorporation from a 4 h pulse with [13C2]acetate into the phytoalexin 2,7-dihydroxycadalene and its methyl ether was measured by direct probe electron impact-mass spectrometry to monitor their biosynthesis at 2, 3, 4, and 6 days after inoculation. Highest incorporation rates occurred during the period of most rapid increase in numbers of fluorescent cells, consistent with the hypothesis that these cells achieve a burst of phytoalexin biosynthesis causing them to become fluorescent as they die. In addition, biosynthesis was still active when fluorescent cell numbers approached their maximum and was appreciably above zero 1 and 2 days later, which suggests the possibility that healthy cells neighboring the hypersensitively responding ones contribute to the biosynthesis of phytoalexins in cotton.