Thiamine treatments alleviate aphid infestations in barley and pea.

Treatment of plants with thiamine (Vitamin B1) has before been shown to activate plant defence against microorganisms. Here, we have studied the effects of thiamine treatments of plants on aphid reproduction and behaviour. The work was mainly carried out with bird cherry-oat aphid (Rhopalosiphum padi L.) on barley (Hordeum vulgare L.). Aphid population growth and aphid acceptance on plants grown from seeds soaked in a 150µM thiamine solution were reduced to ca. 60% of that on control plants. R. padi life span and the total number of offspring were reduced on barley plants treated with thiamine. Healthy aphids and aphids infected with the R. padi virus were similarly affected. Spraying or addition of thiamine at 150µM to nutrient solutions likewise resulted in reduced aphid population growth to ca. 60%, as did plant exposure to thiamine odour at 4mM. Thiamine treatments resulted in reduced aphid population growth also when tested with grain aphid (Sitobion avenae F.) on barley and pea aphid (Acyrthosiphon pisum H.) on pea (Pisum sativum L.). There was no direct effect of thiamine on aphid reproduction or thiamine odour on aphid behaviour, as evaluated using artificial diets and by olfactometer tests, respectively. Two gene sequences regulated by salicylic acid showed higher transcript abundance and one gene sequence regulated by methyl jasmonate showed lower transcript abundance in thiamine-treated plants but not in control plants after aphid infestation. These results suggest that the aphid antibiosis and antixenosis effects may be related to priming of defence, but more studies are needed to explain the effects against aphids.

Differential regulation of 3-aminomethylindole/N-methyl-3-aminomethylindole N-methyltransferase and gramine in barley by both biotic and abiotic stress conditions.

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.

Stronger induction of callose deposition in barley by Russian wheat aphid than bird cherry-oat aphid is not associated with differences in callose synthase or beta-1,3-glucanase transcript abundance.

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.

Microarray analysis of the interaction between the aphid Rhopalosiphum padi and host plants reveals both differences and similarities between susceptible and partially resistant barley lines.

The bird cherry-oat aphid (Rhopalosiphum padi L.) is an important pest on cereals causing plant growth reduction without specific leaf symptoms. Breeding of barley (Hordeum vulgare L.) for R. padi resistance shows that there are several resistance genes, reducing aphid growth. To identify candidate sequences for resistance-related genes, we performed microarray analysis of gene expression after aphid infestation in two susceptible and two partially resistant barley genotypes. One of the four lines is a descendant of two of the other genotypes. There were large differences in gene induction between the four lines, indicating substantial variation in response even between closely related genotypes. Genes induced in aphid-infested tissue were mainly related to defence, primary metabolism and signalling. Only 24 genes were induced in all lines, none of them related to oxidative stress or secondary metabolism. Few genes were down-regulated, with none being common to all four lines. There were differences in aphid-induced gene regulation between resistant and susceptible lines. Results from control plants without aphids also revealed differences in constitutive gene expression between the two types of lines. Candidate sequences for induced and constitutive resistance factors have been identified, among them a proteinase inhibitor, a serine/threonine kinase and several thionins.

N-Methyltransferase involved in gramine biosynthesis in barley: cloning and characterization.

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 microM and 184 microM, 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 MT 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 MT 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.

Subcellular localization of beta-glucosidase in rye, maize and wheat seedlings.

The subcellular compartmentation of beta-glucosidase was studied in rye, maize and wheat seedlings by immunocytochemical methods. For detection, we used a 10 nm gold-labeled secondary antibody, and results were observed using transmission electron microscopy. In all three species, beta-glucosidase was found in plastids, cytoplasm and cell walls. In rye, gold particles were seen on cell walls and cytoplasm in epidermal cells of the root tip and shoot, in bundle sheath cells of the shoot and in all cells, except the vascular bundle cells of the coleoptile. Gold labeling was also observed in plastids of the bundle sheath cells of rye shoot tips and in cortical cells of root tips. In wheat, gold labeling was observed on cell walls and cytoplasm of epidermal cells in the shoot base and coleoptile, and on cell walls and plastids in epidermal cells of the root tip. In maize, gold labeling was mainly found in plastids or proplastids in vascular bundle cells and bundle sheath cells of the shoot, in bundle sheath cells of the coleoptile and in epidermal cells of the root. Some gold particles were also found in cell walls and cytoplasm of stomatal guard cells of the shoot base and vascular bundle cells of the shoot tip and in the cell walls of bundle sheath cells of the shoot tip and root tip epidermal cells. Results are discussed in relation to the role of beta-glucosidase in hydroxamic acid release and overall defense mechanism of monocotyledons.