The expression of NMT (3-aminomethylindole/N-methyl-3-aminomethylindole N-methyltransferase; EC 2.1.1.), involved in the biosynthesis of the indole alkaloid gramine, was investigated in aphid-infested barley (Hordeum vulgare L). NMT is induced by methyl jasmonate and it was hypothesized that the gene would be more strongly upregulated in aphid-resistant barley. We examined the effects of feeding by three aphid species; Russian wheat aphid (Diuraphis noxia Mordvilko), rose-grain aphid (Metopolophium dirhodum Walker) and bird cherry-oat aphid (Rhopalosiphum padi L.) on barley genotypes with varying resistance characteristics. The barley genotypes selected included the cultivar Libra, known to upregulate gramine after feeding by Schizaphis graminum. Infestation by R. padi and M. dirhodum resulted in higher NMT expression in the doubled haploid line 5172-28:4 (DH28:4), which has moderate resistance against R. padi, but not in other aphid barley combinations. None of the aphid plant combinations had however increased gramine, suggesting that aphid-induction of gramine is specific to S. graminum. The increased abundance of NMT transcript in aphid-infested DH28:4 did not lead to higher amounts of NMT protein or NMT enzyme activity, neither did 200 times upregulation of NMT transcript in cotyledons incubated with methyl jasmonate, illustrating that even large differences measured at transcript level may have no metabolic consequences. Drought stress or treatments with abscisic acid did lead to higher gramine concentrations in several barley cultivars, but without any concomitant increase of NMT transcripts. Thus, the regulation of the biosynthetic pathway to gramine at transcript and metabolite level diverges during two different stress conditions.

The effects of infestation by the bird cherry-oat aphid (BCA), (Rhopalosiphum padi L) and the Russian wheat aphid (RWA) (Diuraphis noxia Mordvilko) on callose deposition and transcription of genes related to callose accumulation were investigated in barley (Hordeum vulgare L. cv. Clipper). The BCA, which gives no visible symptoms, induced very limited callose deposition, even after 14 days of infestation. In contrast, RWA, which causes chlorosis, white and yellow streaking and leaf rolling, induced callose accumulation already after 24 h in longitudinal leaf veins. The deposition was pronounced after 72 h, progressing during 7 and 14 days of infestation. In RWA-infested source leaves, callose was also induced in longitudinal veins basipetal to the aphid-infested tissue, whereas in sink leaves, more callose deposition was found above the feeding sites. Eight putative callose synthase genes were identified in a database search, of which seven were expressed in the leaves, but with similar transcript accumulation in control and aphid-infested tissue. Five out of 12 examined beta-1,3-glucanases were expressed in the leaves. All five were upregulated in RWA-infested tissue, but only two in BCA-infested tissue, and to a lesser extent than by RWA. The results suggest that callose accumulation may be partly responsible for the symptoms resulting from RWA infestation and that a callose-inducing signal may be transported in the phloem. Furthermore, it is concluded that the absence of callose deposition in BCA-infested leaves is not because of a stronger upregulation of callose-degrading beta-1,3-glucanases in this tissue, as compared to RWA-infested leaves.

The indole alkaloid gramine occurs in leaves of certain barley (Hordeum vulgare L.) cultivars but not in others. A gene sequence in barley that earlier was characterized as a jasmonate-induced O-methyltransferase (MT) (EC 2.1.1.6, GenBank accession U54767) was here found to be absent in some barley cultivars and breeding lines that all lacked gramine. The cDNA was cloned and expressed in Escherichia coli and the recombinant protein purified. The purified recombinant protein methylated two substrates in the pathway to gramine: 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI) at a high rate, with Km-values of 77 mu M and 184 mu M, respectively. In contrast, the protein did not exhibit any detectable methylation with the earlier suggested substrate for O-methylation, caffeic acid. A number of cultivars and breeding lines of barley were analyzed for presence of the U54767 gene sequence and NIT protein and the enzyme activity in vitro with MAMI or caffeic acid as substrates. The results showed a clear relationship between the presence of the MT gene, the NIT protein and N-methyltransferase activity, and confirmed the identification of the gene as coding for an N-methyltransferase (NMT, EC 2.1.1) and being involved in gramine biosynthesis.