David R. Threlfall

Co-ordinated inhibition of squalene synthetase and induction of enzymes of sesquiterpenoid phytoalexin biosynthesis in cultures of Nicotiana tabacum

Abstract Treatment of log-phase cell suspension cultures of Nicotiana tabacum with the biotic elicitor cellulase completely inhibited cellular growth within a few hours and caused a rapid accumulation of the sesquiterpenoid phytoalexins capsidiol and debneyol. Acetosyringone (an unrelated phenolic compound) was also produced by the elicited cultures. Capsidiol levels fell to low concentrations after the accumulation period had ceased whereas the level of debneyol was maintained and that of acetosyringone was bi-phasic. Growth of the elicited cultures resumed after 24 hr and paralleled that of control cultures. When capsidiol or debneyol were supplied to unelicited cultures they were rapidly catabolized to low levels. Elicitor treatment inhibited the catabolism of capsidiol within three hr but did not affect the metabolism of debneyol. A cell-free preparation obtained from cells 12 hr after elicitation efficiently catalysed the formation of two uncharacterised sesquiterpenoids (one of which [unknown I ] may be an eremophilane hydrocarbon) when incubated with either [1- 14 C]IPP or [1- 3 H 2 ]FPP as substrate. In the presence of NADPH and O 2 , the cell-free system catalysed the formation of capsidiol and debneyol with the amount of unknown I being proportionately reduced. None of the above biosynthetic capabilities were present in cell-free extracts from unelicited cells. The total amount of radioactivity incorporated into capsidiol, debneyol and the two unknowns provided an indirect estimate of the activity of FPP-carbocyclase, the enzyme catalysing the first committed step in the biosynthesis of these compounds. The time course of the rate of synthesis of capsidiol and debneyol in cell-free preparations showed that the synthesis of the phytoalexins was detectable within two hours after elicitation of the cultures with cellulase. Thereafter, the rate of synthesis of both phytoalexins rose rapidly with the maximum rate for each phytoalexin preceding the maximum level of its accumulation. The synthesis of unknowns I and II was detectable at all time points after elicitation. Squalene synthetase (EC 2.5.1.21) activity underwent a rapid decline over the first six hr period of elicitation and remained low until about 48 hr. The apparent K m values (FPP as substrate) for squalene synthetase from unelicited cultures and for capsidiol, debneyol, unknown I and unknown II biosynthesis in elicited cultures were 10, 2, 12, 10 and 10 μm respectively.The rapid inhibition of squalene synthetase in elicited cultures may operate to channel FPP away from sterol biosynthesis (which is required for cellular growth) and towards the FPP-carbocyclase involved in the biosynthesis of the two sesquiterpenoid phytoalexins or may be part of a more general response of plant cells to treatment with elicitors. The levels of debneyol which accumulated in the cultures in response to cellulase were lower than those of capsidiol. These differences are thought to be due to the inhibitory effect of cellulase on the catabolism of capsidiol but not that of debneyol. The results indicate that a co-ordinated sequence of changes involving the regulation of several enzymic activities occurs in tobacco cultures upon elicitation. Studies on the regulation of sterol biosynthesis by elicitor treatment may have wider implications for other eukaryotic systems.

The polysaccharide structure of potato cell walls: Chemical fractionation

Cell walls of potato tubers were fractionated by successive extraction with various reagents. A slightly degraded pectic fraction with 77% galacturonic acid was extracted in hot, oxalate-citrate buffer at pH 4. A further, major pectic fraction with 38% galacturonic acid was extracted in cold 0.1 M Na2CO3 with little apparent degradation. These two pectic fractions together made up 52% of the cell wall. Most of the oxalate-citrate fraction could alternatively be extracted with cold acetate-N,N′,N′-tetracetic acid (CDTA) buffer, a non-degradative extractant which nevertheless removed essentially all the calcium ions. This fraction was therefore probably held only by calcium binding, and the remainder of the pectins by covalent bonds. Electrophoresis showed that both pectic fractions contained a range of molecular types differing in composition, with a high arabinose: galactose ratio as well as much galacturonic acid in the most extractable fractions. From methylation data, the main side-chains were 1,4′-linked galactans and 1,5′-linked arabinans, with smaller quantities of covalently attached xyloglucan. Extraction with NaOH-borate removed a small hemicellulose fraction and some cellulose. The main hemicelluloses were apparently a galactoxyloglucan, a mannan or glucomannan and an arabinogalactan.

5-epi-aristolochene is a common precursor of the sesquiterpenoid phytoalexins capsidiol and debneyol

Abstract 5- epi -Aristolochene (4- epi -eremophila-9, 11-diene) has been identified (GC-MS and high-field 1 H NMR) as the hydrocarbon (unknown I ) which is accumulated when the formation of capsidiol and debneyol from [1- 3 H 2 ]FPP in cell-free preparations of cells from cellulase-elicited cell-suspension cultures of Nicotiana tabacum is inhibited by the omission of NADPH or the exclusion of molecular oxygen from the incubation mixture. Feeding experiments with 14 C-labelled 5- epi -aristolochene have shown this compound to be a common precursor of the two sesquiterpenoid phytoalexins capsidiol and debneyol which accumulate in the elicited cultures. The evidence suggests that the biosynthesis of capsidiol is regulated, in part, by the activity of the first of the two hydroxylases which catalyse its formation from this hydrocarbon and furthermore, that this 3-hydroxylase is induced during the elicitation process.

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 (R1) 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.

Abiotic factors elicit sesquiterpenoid phytoalexin production but not alkaloid production in transformed root cultures of Datura stramonium

The treatment of root cultures of Datura stramonium with copper and cadmium salts at external concentrations of approximately 1mM has been found to induce the rapid accumulation of high levels of sesquiterpenoid defensive compounds, notably lubimin and 3-hydroxylubimin. These compounds were undetectable in unelicited cultures. No net change was seen in the alkaloid content of the system following treatment with Cu2+ or Cd2+, the tropane alkaloid titre apparently being insensitive to elicitation. However, a considerable rapid and, in some instances, reversible release of alkaloid was observed. This resulted in the appearance of up to 50–75% of the total alkaloid in the medium after 40–60 h. Subsequently, in cultures treated with Cu2+ ions, though not in cultures treated with Cd2+ ions, this alkaloid was re-absorbed. These observations show how, in a single system, different groups of secondary products can show distinct differences in their responses to potential elicitors.

Regulation and enzymology of pentacyclic triterpenoid phytoalexin biosynthesis in cell suspension cultures of Tabernaemontana divaricata

Abstract Treatment of growth-phase cell suspension cultures of Tabernaemontana divaricata with a Candida albicans elicitor preparation inhibited both growth and monoterpene indole alkaloid production within a few hours and caused a rapid accumulation of at least 10 pentacyclic triterpenoid phytoalexins. Cell growth was resumed at about the same time (36hr after elicitor treatment) as phytoalexin accumulation ceased. Alkaloid production, however, was not resumed until some 72 hr after elicitation. Cell-free preparations obtained from cells 12 and 24 hr after elicitation efficiently catalysed the synthesis of squalene, squalene 2,3-oxide, α- and β-amyrin, uvaal and oleanal, ursolic and oleanolic acid, some monohydroxy derivatives of these acids, and dihydroxy derivatives of 3- epi -ifflaconic acid when incubated with either [1- 14 C]IPP or [1- 3 H 2 ]FPP as substrate, Preparations from unelicited cells synthesized squalene and small amounts of cycloartenol only. Squalene synthetase (EC 2.5.1.21 activity increased rapidly by approximately five-fold within 24 hr in response to the elicitor treatment and then declined slowly to basal levels. Feeding experiments with saturating levels (for sterol synthesis) of ( R )-[2- 14 C]MVA indicated that in elicited cultures the synthesis of phytosterols (and hence growth) was inhibited at the level of squalene 2,3-oxide: cycloartenol cyclase.

Cell wall polysaccharides from onions

Abstract Onion ( Allium cepa ) cell walls were fractionated by successive extraction with oxalate-citrate buffer and with alkali. The substantial oxalate-citrate extracted fraction comprised a range of pectic polysaccharides with varying proportions of neutral side-chains. Methylation analysis of the alkali extract indicated that (1,4′)-linked galactans and a substituted xyloglucan were probably major components. Onions thus resemble dicotyledonous plants more than the Gramineae in their cell wall composition.

Biosynthesis of tocopherols: A re-examination of the biosynthesis and metabolism of 2-methyl-6-phytyl-1,4-benzoquinol

Abstract A number of previous studies of the involvement of 2-methyl-6-phytyl-1,4-benzoquinol in the biosynthesis of α-tocopherol have failed to take account of the fact that this quinol and its quinone have very similar chromatographic properties to those of 2-methyl-3-phytyl-1,4-benzoquinol and 2-methyl-3-phytyl-1,4-benzoquinone respectively. It has now been shown that the two quinones can be separated from each other either by multidevelopment TLC or by HPLC and that the claims made earlier with regard to the biosynthesis and metabolism of 2-methyl-6-phytyl-1,4-benzoquinol in chloroplasts are correct. In particular, it has been established that this quinol is the only methyl phytylbenzoquinol formed from homogentisate and phytyl pyrophosphate in chloroplast preparations. It has also been shown for the first time that lettuce chloroplasts are able to synthesize 3 H-labelled α- and γ-tocopherols from [ methylene - 3 H] homogentisate.

Accumulation of phytoalexins in potato-cell suspension cultures

Abstract Potato-cell suspension cultures (ex Kennebec tuber tissue) accumulate the sesquiterpenoid phytoalexins lubimin, rishitin and solavetivone after inoculation with sporangia of either a compatible or an incompatible race of the fungal pathogen Phytophthora infestans . The phytoalexins are present in the cells and the medium. No evidence was obtained for the formation of any metabolites of rishitin.

Enzymological aspects of the redirection of terpenoid biosynthesis in elicitor-treated cultures of Tabernaemontana divaricata

Abstract The elicitor-mediated induction of pentacyclic triterpenoid phytoalexin accumulation in cells of five-day-old suspension cultures of Tabernaemontana divaricata is accompanied by: a rapid and transient increase in HMG-CoA reductase (EC 1.1.1.34) activity; an increase in IPP isomerase (EC 5.3.3.2), prenyl transferase (EC 2.5.1.1) and squalene synthetase (EC 2.5.1.21) activity; a rapid inhibition of squalene 2,3-oxide:cycloartenol cyclase activity (EC 5.4.99.8), and a rapid and relatively transient appearance of squalene 2,3-oxide:amyrin cyclase (EC 5.4.99.-) activity. These findings are entirely consistent with an elicitor-induced redirection of the cytosolic-microsomal pathway of terpenoid biosynthesis away from phytosterol biosynthesis and towards pentacyclic triterpenoid phytoalexin biosynthesis. The switch being mediated as a direct result of the rapid inhibition of squalene 2,3-oxide:cycloartenol cyclase activity just prior to the de novo synthesis of squalene 2,3-oxide:amyrin cyclase and the other enzymes on the post-squalene 2,3-oxide span of the pentacyclic triterpenoid phytoalexin pathway. The increased activities of the enzymes common to both pathways reflects the fact that the rate of accumulation of pentacyclic triterpenoid phytoalexins in elicited cultures is more rapid than the rate of phytosterol biosynthesis in control cultures. The very rapid and transient increase in HMG-CoA reductase activity points to the microsomal form(s) of this enzyme having a key regulatory role in controlling the flux of carbon into the cytosolic-microsomal pathway of terpenoid biosynthesis.