Biosynthesis and metabolism of sesquiterpenoid phytoalexins and triterpenoids in potato cell suspension cultures

Abstract

The accumulation and turnover of sesquiterpenoid phytoalexins and the effects of the induction of phytoalexin biosynthesis on triterpenoid synthesis have been studied in potato tuber tissue discs and cell suspension cultures inoculated with sporangia of either an incompatible (Race 4) or compatible (Complex) race of Phytophthora infestans. A comparative study of the incompatible and compatible interaction using Kennebec (R 1) tuber tissue showed the accumulation of rishitin (major component) and lubimin in both interations, though the patterns of accumulation and the observed fungal development were different. Rishitin turnover in both interactions was demonstrated by administration early in the time course of a small dose of [2- 14C]mevalonate though no accumulation of a rishitin metabolite was apparent. The assay of the incorporation over a series of short time periods (2 hr) of either [2- 14C]MVA or [2- 14C]acetate into squalene and sterols suggested that triterpenoid synthesis by tuber discs ( P. infestans is unable to synthesize sterols) was inhibited during the period of phytoalexin accumulation. A similar study with Kennebec-cell suspension cultures showed the accumulation of rishitin, mostly in the growth medium, when the cultures were inoculated with sporangia of P. infestans. Unlike the tuber discs, however, the patterns of accumulation of rishitin were identical with both races of fungus and the zoospores liberated from the sporangia did not germinate and infect the potato cells. A marked loss of rishitin from the inoculated culture occurred after 24 hr, but, as with tuber discs, this loss took place without the accumulation of any rishitin metabolise. As judged by the incorporation over short time periods (2 hr) of 12- 14C]MVA into squalene, sterols and phytoalexins, triterpenoid synthesis was markedly reduced just prior to the onset of phytoalexin accumulation. Potato cv. Majestic (r)-cell suspension cultures inoculated with sporangia of P. infestans Complex race accumulated lubimin, 3-hydroxylubimin, rishitin, phytuberol and phytuberin in the growth medium. The patterns of accumulation of phytoalexins in the presence or absence of a saturating concentration for sterol synthesis of either [2- 14C]MVA (3.3 mM) or 12- 14C]acetate (1 mM) were in agreement with the partial biosynthetic sequence lubimin → 3-hydroxylubimin → rishitin. Triterpenoid synthesis, as measured by the incorporation of the 3.3 mM [2- 14C]MVA and 1 mM [2- 14C]acetate, ceased very abruptly just prior to the start of phytoalexin accumulation. The reason for this appeared to be due to the inhibition or loss of squalene synthetase activity. The presence of 3.3 mM [2- 14C]MVA in the cultures resulted in a large increase in the levels of squalene, squalene 2,3-oxide and cycloartenol in the cells of healthy cultures and of phytoalexins accumulated in the growth medium of the inoculated cultures. This apparent lack of any regulatory control of the incorporation of MVA into the three triterpenoids and into phytoalexins is presumably the principal reason for the need to bring about the rapid and complete inhibition of squalene synthetase activity in the cells of inoculated cultures and, by implication, in the cells responsible for the synthesis of phytoalexins in infected tuber discs.