Gramine Accumulation in Leaves of Barley Grown under High-Temperature Stress

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 (N,N-dimethyl-3-aminomethylindole) and lesser amounts of its precursors 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI); other cultivars such as ;Proctor' do not. When grown at optimal temperatures (21 degrees C/16 degrees 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 degrees C/16 degrees C to supra-optimal temperatures (>/=30 degrees C/25 degrees C), 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 -->--> AMI --> MAMI --> gramine) between tryptophan and AMI. (a) Proctor and Arimar leaves readily absorbed [(14)C]gramine, but neither cultivar degraded it extensively. (b) Arimar leaf tissue incorporated [(14)C]formate label into the N-methyl groups of gramine and MAMI, and converted [methylene-(14)C]tryptophan to AMI, MAMI, and gramine; Proctor leaf tissue did not, even when a trapping pool of unlabeled gramine was supplied. (c) Proctor converted [(14)C]MAMI to gramine as actively as Arimar. (d) Proctor incorporated [(14)C]formate label into gramine and MAMI when supplied with AMI; the ratio [(14)C]gramine/[(14)C]MAMI 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, plantlet growth was reduced by gramine although callus growth was not. We speculate that gramine-accumulating cultivars may suffer autotoxic effects at high leaf temperatures.