A C-13-PULSE-LABELING STUDY OF PHYTOALEXIN BIOSYNTHESIS IN HYPERSENSITIVELY RESPONDING COTTON COTYLEDONS

Time courses of multiplication of an incompatible strain of Xanthomona s campestris pv. malvacearum accumulation of phytoalexins, and develop ment of phytoalexin-rich, yellow-green fluorescent, hypersensitively n ecrotic cells were followed in cotton cotyledons. Occasionally, hypers ensitively responding palisade cells were seen to pass through a stage in which the yellow-green fluorescence, due to the phytoalexins lacin ilene C and its methyl ether, was confined to the cytoplasmic layer su rrounding 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 pos t-inoculation, was also the period of most rapid increase in phytoalex in content, and the rate of bacterial multiplication began to slow dow n at this lime. Stable C-13 isotope incorporation from a 4h pulse with [C-13(2)]acetate into the phytoalexin 2,7-dihydroxycadalene and its m ethyl ether was measured by direct probe electron impact-mass spectrom etry to monitor their biosynthesis at 2, 3, 4, and 6 days after inocul ation. Highest incorporation rates occurred during the period of most rapid increase in numbers of fluorescent cells, consistent with the hy pothesis that these cells achieve a burst of phytoalexin biosynthesis causing them to become fluorescent as they die. In addition, biosynthe sis was still active when fluorescent cell numbers approached their ma ximum and was appreciably above zero 1 and 2 days later, which suggest s the possibility that healthy cells neighboring the hypersensitively responding ones contribute to the biosynthesis of phytoalexins in cott on. (C) 1995 Academic Press Limited