The indole alkaloid gramine is toxic to animals and may play a defensive role in plants. Under certain conditions, shoots of barley cultivars such as 'Arimar' and CI 12020 accumulate gramine (NN-dimethyl-3-aminomethylindole) and lesser amounts of its precursors 3-aminomethylindole (AMI) and N-methyl-3-amithyllndole (MAMI); other cultivars such as 'Proctor' do not. When grown at optimal temperatures (21°C/16°C, day/ night), Arimar contained a high level of gramine in the first leaf (approximately 6 milligrams per gram dry weight), but progressively less accumulated in successive leaves so that the gramine level in the shoot as a whole fell sharply with age. In Arimar and CI 12020 plants transferred at the two-to three-leaf stage from 21°C/16°C to supra-optimal temperatures (:300C/250C), there was massive gramine accumulation in leaves which developed at high temperature, so that gramine level in the whole shoot remained high (about 3-8 milligrams per gram dry weight).Proctor lacked both constitutive gramine accumulation in the first leaf and heat-induced gramine accumulation in later leaves. The following evidence indicates that this results from a lesion in the pathway of synthesis (tryptophan - MAMI when supplied with AMI; the ratio 114Cjgramine/i14CIMAMI fell with leaf age, suggesting that the two N-methylations involve different enzymes. Inasmuch as Proctor leaf tissue did not methylate added tryptamine or tyramine, the N-methyltransferase(s) of gramine synthesis may be substrate specific.-In sterile culture at optimal temperatures, 10 millimolar gramine did not affect autotrophic growth of Arimar or Proctor plantlets or heterotrophic growth of callus. At supra-optimal temperature, plantiet growth was reduced by gramine although callus growth was not. We speculate that gramine-accumulating cultivars may suffer autotoxic effects at high leaf temperatures.The simple indole alkaloid gramine (N,N-dimethyl-3-aminomethylindole) is a well-known constituent of young shoots of certain barley (Hordeum vulgare L.) cultivars where it can reach concentrations of about 8 mg g-' dry weight at the one-to threeleaf stage (e.g. 3, 9, 23, 24 other indolealkylamines can occur also in foliage of the forage grass Phalaris arundinacea L. (reed canarygrass); in this crop, the indole alkaloids are important anti-quality components because they are toxic to grazing animals and adversely affect their health and weight gain (17). Gramine has been shown also to have phytotoxic effects on chickweed (22). Although in reed canarygrass it is established that the levels of gramine and other indole alkaloids are subject to both genetic (18) and environmental (15, 16) control, little is known of genetic or environmental regulation of gramine in barley (12). Correspondingly little is known for barley about the physiological, ecological, and agronomic significance of gramine accumulation. Roles as a feeding deterrent for herbivores (1), as a nematotoxin (3), and as an allelopathic substance (22) have been suggested. Note that all these...

No abstract

Previous work showed that the indole alkaloid gramine accumulates in the upper leaves (e.g. the fifth) of barley as a response to high growth temperatures. The biosynthesis of gramine proceeds from tryptophan to 3-aminomethylindole (AMI); sequential N-methylations of AMI then yield N-methyl-3-aminomethylindole (MAMI) and gramine.To determine whether high-temperature stress increases the activity of gramine pathway enzymes, leaf tissue from plants grown at various temperatures was assayed for N-methyltransferase (NMT) activity using AMI and MAMI as substrates in both in vivo and in vitro assays. NMT activity in expanding fifth leaves was increased 8-to 20-fold by growth at high temperatures (35°C day/30°C night) compared to cool temperatures (15°C/10°C). Several days of high temperature were required for full induction of NMT activity. No induction of NMT activity occurred in leaves which had completed expansion in cool conditions before exposure to high temperature.To investigate NMT induction at the protein level, NMT activity was purified to homogeneity and used to produce polyclonal antibodies. Throughout enzyme purification, relative NMT activities towards AMI and MAMI remained constant, consistent with a single NMT enzyme. Immunoblot analysis showed that a large increase in NMT polypeptide coincided with induction of NMT activity by heat stress. Our results point to a type of high-temperature regulation of gene expression that is quite distinct from heat shock.Gramine is a simple indole alkaloid found in the shoots of many barley (Hordeum vulgare L.) cultivars and wild barley lines (3,12). Gramine biosynthesis involves the steps shown in Scheme 1. The indole nucleus and the methylene side chain oftryptophan are incorporated into the first stable intermediate ofthe pathway, AMI3 (7,9). AMI is then methylated at the amino nitrogen to form the secondary amine, MAMI, which is in turn N-methylated to produce the tertiary amine, gramine (7,18,22). Indirect evidence indicates that these methylations are catalyzed by an NMT enzyme (or enzymes) specific to the gramine pathway (1 1), for which SAM acts as the methyl donor (18). Although degradative pathways for gramine are known (6), gramine catabolism is very slow (1 1) so that accumulation is controlled mainly by ' Research conducted under contract DE-AC02-76ERO-1 338 from the United States Department of Energy. Michigan Agricultural Experimental Station Journal Article 11630. 2Present address: Funk Seeds International,