Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore.

Herbivory induces both direct and indirect defenses in plants; however, some combinations of these defenses may not be compatible. The jasmonate signal cascade activated both direct (nicotine accumulations) and indirect (mono- and sesquiterpene emissions) whole-plant defense responses in the native tobacco Nicotiana attenuata Torr. Ex Wats. Nicotine accumulations were proportional to the amount of leaf wounding and the resulting increases in jasmonic acid (JA) concentrations. However, when larvae of the nicotine-tolerant herbivore, Manduca sexta, fed on plants or their oral secretions were applied to leaf punctures, the normal wound response was dramatically altered, as evidenced by large (4- to 10-fold) increases in the release of (i) volatile terpenoids and (ii) ethylene, (iii) increased (4- to 30-fold) accumulations of endogenous JA pools, but (iv) decreased or unchanged nicotine accumulations. The ethylene release, which was insensitive to inhibitors of induced JA accumulation, was sufficient to account for the attenuated nicotine response. Applications of ethylene and ethephon suppressed the induced nicotine response and pre-treatment of plants with a competitive inhibitor of ethylene receptors, 1-methylcyclopropene, restored the full nicotine response. This ethylene burst, however, did not inhibit the release of volatile terpenoids. Because parasitoids of Manduca larvae are sensitive to the dietary intake of nicotine by their hosts, this ethylene-mediated switching from direct to a putative indirect defense may represent an adaptive tailoring of a plant's defense response.

Auxins induce tryptophan decarboxylase activity in radicles of catharanthus seedlings.

Germinating seedlings of Catharanthus roseus produce monoterpenoid indole alkaloids as a result of a transient increase of tryptophan decarboxylase (TDC) activity. The influence of auxins on this transient rise of TDC activity was studied. External application of indolebutyric acid or 2,4-dichlorophenoxyacetic acid at a concentration of 20 to 40 mum enhanced and prolonged the rise in TDC activity in developing seedlings. Auxin treatment also influenced the morphology of the seedlings; it induced a shortening and thickening of the hypocotyl and the radicle and promoted the initiation of lateral roots in the radicle. During development, the radicles of auxin-treated seedlings displayed a gradual increase in TDC activity that was absent in the radicles of untreated controls. Examination of immunoblots revealed anti-TDC reactive proteins in extracts from radicles of auxin-treated seedlings, but none in extracts from radicles of control seedlings. In contrast, TDC activity and immunoreactive protein levels in the aerial parts of controls and auxin-treated seedlings were comparable. Our results indicate that externally applied auxins induce both abnormal development and TDC activity in the radicles of Catharanthus seedlings. Although auxins slightly delayed the light-mediated induction of the cotyledon-specific last step in vindoline biosynthesis (i.e. acetylcoenzyme A: deacetylvindolin-O-acetyltransferase activity), seedlings still synthesized vindoline, one of the major alkaloid end products.

Phytochrome is involved in the light-regulation of vindoline biosynthesis in catharanthus.

The enzyme acetylcoenzyme A:deacetylvindoline 4-O-acetyl-transferase (DAT) catalyzes the final step in the biosynthesis of the monoterpenoid indole alkaloid, vindoline. Previous studies have shown that the appearance of DAT activity in etiolated seedlings of Catharanthus roseus is induced by exposure of seedlings to light and that enzyme activity is restricted principally to the cotyledons. Evidence is now presented that phytochrome is involved in the light-mediated induction of DAT activity in Catharanthus cotyledons.

The influence of tryptophan and tryptamine feeding and light on alkaloid biosynthesis in cinchona seedlings.

It has previously been shown that, at the onset of germination of CINCHONA seeds, transient increases in both the tryptophan level and tryptophan decarboxylase (TDC) activity occur. Subsequently, in the seedlings the levels of the TDC product tryptamine and its derived alkaloids increase. We investigated whether this process can be influenced by external tryptophan or tryptamine feeding. Besides, the possible role of light in alkaloid biosynthesis was studied by monitoring this process in etiolated seedlings. Tryptophan feeding slightly raised the TDC activity in the seedlings, but alkaloid production remained unaltered. During tryptamine feeding, the transient increases in tryptophan level and TDC activity were still observed, and also here, alkaloid production remained unaltered. Finally, in etiolated seedlings the alkaloid biosynthetic pathway proceeded normally. Thus, in germinating CINCHONA seedlings alkaloid production is not susceptible to tryptophan or tryptamine feeding and independent of light.

Detrimental effects ofCinchona leaf alkaloids on larvae of the polyphagous insectSpodoptera exigua.

YoungCinchona ledgeriana plants contain two types of alkaloid: indole alkaloids in the leaves and quinoline alkaloids in the root. FromCinchona leaves, a crude alkaloid extract was made, containing the cinchophylline type of indole alkaloids and a small amount of 5-methoxytryptamine. The leaf alkaloid extract exerted a strong detrimental effect on the growth of larvae of the polyphagous beet armyworm,Spodoptera exigua (Lepidoptera). Feeding of larvae on an artificial diet containing the leaf alkaloids at the same concentrations as those found in the plant resulted in significant growth reduction, retardation in development, and mortality of the larvae. Cinchophyllines are composed of 5-methoxytryptamine coupled to a corynantheal unit. When incorporated into the artificial diet, 5-methoxytryptamine alone had no effect on the 5.exigua larvae. Corynantheal, however, had a strong detrimental effect on growth of the larvae, its effect being comparable to that of the leaf alkaloid extract. In contrast to the indole-type leaf alkaloids, the quinolinetypeCinchona root alkaloids did not affect growth and development of the larvae. These results suggest that the indole-type alkaloids, which inCinchona plants are present at the highest concentrations in the young, vulnerable leaflets, are involved in the chemical defense of the plant against herbivorous insects.

The distribution of strictosidine-synthase activity and alkaloids in Cinchona plants.

The relation between the total alkaloid content and the activity of strictosidine synthase (EC 4.3.3.2), a key enzyme in alkaloid biosynthesis, was studied in distinct parts of six-month-old plants of Cinchona ledgeriana Moens. Strictosidine-synthase activity was present in the tops of the stems, including the young developing leaflets, and in the roots. The highest alkaloid contents of the plant were also found in these parts; however, the types of alkaloids differed, cinchophyllines being present in the aerial parts and quinoline alkaloids in the roots. In the stem and in old leaves, both strictosidine-synthase activity and alkaloid content were low. These results indicate that in young Cinchona plants the alkaloids are mainly synthesized in the axial extremities of the plant and that they are stored at the site of their synthesis.

Single-chain and two-chain tissue-type plasminogen activator (t-PA) bind differently to cultured human endothelial cells.

It has recently been shown that the fibrinolytic components plasminogen and tissue-type plasminogen activator (t-PA) both bind to cultured human umbilical vein endothelial cells (HUVEC). After cleavage of t-PA by plasmin, "single-chain" t-PA (sct-PA) is converted into "two-chain" t-PA (tct-PA), which differs from the former in a number of respects. We compared binding of sct-PA and tct-PA to the surface of HUVEC. Removal of t-PA bound to HUVEC by a mild treatment with acid and a subsequent quantification of eluted t-PA both by activity- and immunoradiometric assays revealed that, at concentrations between 10 and 500 nM, HUVEC bind about 3-4 times more sct-PA than tct-PA. At these concentrations, both sct-PA and tct-PA remain active when bound to HUVEC. Mutual competition experiments showed that sct-PA and tct-PA can virtually fully inhibit binding of each other to HUVEC, but that an about twofold higher concentration of tct-PA is required to prevent half-maximal binding of sct-PA than visa versa. These results demonstrate that sct-PA and tct-PA bind with different affinities to the same binding sites on HUVEC.

Cyclic-AMP-induced elevation of intracellular pH precedes, but does not mediate, the induction of prespore differentiation in Dictyostelium discoideum.

Prespore gene expression in Dictyostelium is induced by the interaction of cAMP with cell surface cAMP receptors. We investigated whether intracellular pH (pHi) changes mediate the induction of prespore gene expression by cAMP. It was found that cAMP induces a 0.15 unit increase in pHi within 45 min after stimulation. The cAMP-induced pHi increase precedes the induction of prespore gene expression, measured by in vitro transcription, by about 15-30 min. Cyclic-AMP-induced pHi changes can be bypassed or clamped by addition of, respectively, the weak base methylamine, which increases pHi, or the weak acid 5,5-dimethyl-2,4-oxazolidinedione (DMO), which decreases pHi. Bypass of the cAMP-induced increase of pHi with methylamine does not induce the expression of prespore genes, while inhibition of the pHi increase with DMO does not inhibit the induction of prespore gene expression. Cyclic-AMP-induced prespore protein synthesis and the proportion of prespore cells in multicellular aggregates are also not affected by bypassing or inhibiting the cAMP-induced pHi increase. These results show that although a morphogen-induced pHi increase precedes the induction of prespore gene expression, this increase does not mediate the effects of the extracellular cAMP signal on the transcription or translation of prespore genes in Dictyostelium discoideum.

Changes in cytoplasmic pH are involved in the cell type regulation of Dictyostelium.

The cytoplasmic pH (pHi) of populations of developing Dictyostelium discoideum cells was determined by means of two independent pH null-point methods. Both methods reveal in populations containing 75-80% prespore cells a pHi value of about 0.2 pH units higher than in populations containing 50% prespore cells. During the process of cell type regulation, decreases and increases in the percentage of prespore cells of about 15-20% are accompanied by decreases and increases in pHi of about 0.2 pH units. Abolition of these changes in pHi by means of a weak base or acid also prevents the regulation process. It is concluded that changes in pHi are involved in the prespore cell type regulation in D. discoideum.

Cell cycle phase in Dictyostelium discoideum is correlated with the expression of cyclic AMP production, detection, and degradation. Involvement of cyclic AMP signaling in cell sorting.

Cell cycle phase in Dictyostelium is correlated with a different preference for either spore or stalk differentiation. Cells which start development early in the cell cycle (E cells) exhibit a strong tendency to sort to the prestalk region of slugs, while late cell cycle cells (L cells) sort to the prespore region. We investigated the expression of the cAMP chemotactic system during development of synchronized E and L cells and found that E cells exhibit cAMP-binding activity, cell surface cAMP-phosphodiesterase (mPDE) activity, and the ability to relay cAMP signals at least 2 hr earlier and to higher levels than L cells. We hypothesize that E cells are prestalk sorters because they are the first to initiate aggregation centers and respond most effectively with chemotaxis and signal relay.

Cyclic AMP induces a transient alkalinization in Dictyostelium.

In a wide range of cell types, stimulus-response coupling is accompanied by a rise in cytoplasmic pH (pHi). It is shown that stimulation of developing Dictyostelium discoideum cells with the chemoattractant cAMP also results in a rise in pHi. About 1.5 min after stimulation, pHi starts increasing from pHi approximately 7.45 to pHi approximately 7.60, as is revealed independently by two different pH null-point methods. The rise in pHi is transient, also with a persistent stimulus, and effectively inhibited by diethylstilbestrol (DES), strongly suggesting that the rise in pHi is accomplished by the DES-sensitive plasma membrane proton pump which has been demonstrated in D. discoideum.

Cytoplasmic pH at the onset of development in Dictyostelium.

Cytoplasmic pH (pHi) was measured by a pH 'null point' method in synchronous populations of Dictyostelium discoideum cells. Synchronous populations of vegetative cells with a lower pHi than asynchronous populations of vegetative cells show a clear preference for the prespore differentiation pathway when they enter development. Treatment of these cells before development with the weak base ammonia results in an increase in pHi and an abolition of the preference of the cells for the prespore pathway. On the other hand, synchronous populations of vegetative cells with the same pHi as asynchronous populations of vegetative cells resemble these last cells in that they show no preference for a certain differentiation direction when they enter development. However, both these synchronous and asynchronous populations of cells do acquire a preference for the prespore pathway when they are treated before development with the weak acid 5,5-dimethyl-2,4-oxazolidineolione (DMO), which lowers their pHi. It is concluded that changes in pHi at the onset of the developmental program lead to significant changes in the preference of the cells for the prespore differentiation pathway in Dictyostelium discoideum.

The epidermal growth factor-induced calcium signal in A431 cells.

Addition of epidermal growth factor (EGF) to human A431 cells causes a 2-4-fold increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured by quin-2 fluorescence. The EGF effect is rapid but transient: [Ca2+]i reaches a maximum within 30-60 s and then returns to its resting value (182 +/- 3 nM) over a 5-8-min period. The EGF-induced [Ca2+]i rise is completely dependent on extracellular Ca2+, is abolished by La3+ and Mn2+, and is not accompanied by changes in membrane potential (mean values of -64 mV). Serum also elicits a transient [Ca2+]i rise in A431 cells, but this response is not dependent on the presence of extracellular Ca2+. The tumor promoter 12-O-tetradecanoylphorbol 13-acetate completely inhibits the EGF- and serum-induced increases in [Ca2+]i without affecting basal [Ca2+]i levels. Our results, together with previous 45Ca2+ uptake data (Sawyer, S. T., and Cohen, S. (1981) Biochemistry 20, 6280-6286), suggest that while serum factors trigger the release of Ca2+ from internal stores, EGF acts by opening a voltage-independent Ca2+ channel in the plasma membrane. The data further suggest a role for protein kinase C in attenuating the Ca2+-mobilizing mechanisms of EGF and serum.

Cytoplasmic pH and the regulation of the Dictyostelium cell cycle.

Cytoplasmic pH (pHi) was monitored during the cell cycle of synchronous populations of Dictyostelium discoideum by means of a pH "null point" method. There is a cycle of pHi that closely corresponds to the DNA replication cycle, with a minimum of pH 7.20 in interphase and a peak of pH 7.45 during S phase and mitosis. When DNA replication is blocked by hydroxyurea, pHi continues to oscillate with a similar period as the uninhibited division cycle. Even when protein synthesis is inhibited by cycloheximide the periodic pHi cycles persist. Artificially raising pHi in exponentially growing cells by approximately 0.1 units causes a severalfold increase in the rates of protein and DNA synthesis. We conclude that an autonomous pHi oscillator exists in Dictyostelium cells, which operates independently of protein and DNA synthesis and which may have a major role in the timing and regulation of the cell cycle.