Biomass (a) and fraction of biomass in roots (b) of <i>Arabidopsis thaliana</i> grown on either NO₃⁻ or on NH₄NO₃ or on different combinations of glutamine and NO₃⁻.

<p>All media had a total N concentration of 6 mM. Plants were grown on sterile agar plates for 21 days. Bars represent average values ± SE, n = 8. Different lower-case letters indicate differences at p≤0.05 between N treatments.</p

Split-root experiment with <i>Arabidopsis thaliana</i>.

<p>Plants were grown on agar plates that were divided into two identical compartments by a plastic rib. The growth medium was identical on both sides of the rib and with N supplied as a mixture of 1.5 mM glutamine+3 mM NO₃⁻ but on one side, one of the N sources (either glutamine or NO₃⁻) was ¹⁵N-labelled. Bars indicate the fraction of N derived from each source and represent average ± SE, n = 6–7. Different lower-case and capital letters indicate differences at p≤0.05 between plants parts and between N sources, respectively.</p

Origin of root N, shoot N and plant N, in <i>Arabidopsis thaliana</i> plants grown on 3 mM NH₄NO₃ (a) or a mixture of 1.5 mM glutamine+3 mM NO₃⁻ (b).

<p>Fractions of N derived from individual N sources in the mixtures were calculated from N contents and rates of ¹⁵N abundance in plant parts. Plants were grown on sterile agar plates for 21 days. Bars represent average values ± SE, n = 5. Different lower-case and capital letters indicate differences at p≤0.05 between plant parts and between N sources, respectively.</p

Split-root experiment with <i>Arabidopsis thaliana</i>.

<p>Plants were grown on agar plates that were divided into two identical compartments by a plastic rib. The two compartments contained either 1.5 mM glutamine or 3 mM NO₃⁻ as N sources. For each plate, one of the N sources (either glutamine or NO₃⁻) was ¹⁵N-labelled. Bars indicate the fraction of N derived from each source for the shoot and for roots growing in the NO₃⁻ compartment and the glutamine compartment. Bars represent average ± SE, n = 5. Different lower-case and capital letters indicate differences at p≤0.05 between plant parts, and between N sources, respectively.</p

Biomass (mg plant⁻¹), N concentrations (mg g⁻¹) and root mass fraction of plants grown in sterile agar culture and with N supplied as either a mixture of 1.5 mM glutamine and 3 mM NO₃⁻ or 3 mM NH₄NO₃.

<p>Average values ± SE, n = 5. Different letters indicate differences at p≤0.05 between N treatments.</p

Long day and night phloem-feeding times are important for successful ¹⁵N uptake by aphids.

<p>The µg excess ¹⁵N uptake of individual <i>R. padi</i> aphids (N = 22) are plotted against the time aphids had been feeding in the phloem phase (E2) on barley plants either during night (blue stars), day (orange stars) or total (day and night; dark green stars). Day (16 hours) and night (8 hours) values of phloem-feeding are extracted out of the total aphid phloem-feeding duration (E2). The feeding experiment was conducted for up to 24 hours and the feeding behaviour was recorded with the EPG method. The µg excess ¹⁵N uptake of control aphids was zero.</p

Stable ¹⁵N enrichment of aphids correlates with phloem-feeding duration.

<p>The calculated values of the µg excess ¹⁵N uptake of <i>R. padi</i> aphids (N = 30) are plotted against the time that aphids spent in defined EPG phases on barley plants. Either the phloem-feeding phase E2 (A) or the sum of defined non-feeding phases are plotted (B). The non-feeding time includes the time aphids spent cell puncturing (C), had derailed stylet mechanics (F), were xylem drinking (G) and saliva injecting (E1). Pearson's product moment correlation between ¹⁵N uptake and phloem-feeding A, t = 8.95, cor = 0.86, p<0.001 and ¹⁵N uptake and non-feeding B, t = −2.03, cor = −0.36, p = 0.05 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083085#pone-0083085-t002" target="_blank">Table 2</a>). The feeding experiment was conducted for up to 24 hours and the feeding behaviour was recorded with EPG. The µg excess ¹⁵N uptake of control aphids was zero.</p

High ¹⁵N enrichment is characterised by few feeding interruptions.

<p>The µg excess ¹⁵N uptake of individual <i>R. padi</i> aphids is plotted against the number of feeding interruptions. Green stars (N = 21), experimental time up to 24 hours. Orange stars (N = 8), experimental time from 4 to 6 hours. A zero value of no feeding behaviour is excluded in the plot and the statistical analyses. Pearson's product moment correlation for 20 hours, t₍₂₀₎ = −3.02, cor = −0.56, p = 0.007; for 4 to 6 hours, t₍₆₎ = −0.73, cor = −0.28, p = 0.495.</p

Exploring the Nitrogen Ingestion of Aphids — A New Method Using Electrical Penetration Graph and ¹⁵N Labelling

<div><p>Studying plant-aphid interactions is challenging as aphid feeding is a complex process hidden in the plant tissue. Here we propose a combination of two well established methods to study nutrient acquisition by aphids focusing on the uptake of isotopically labelled nitrogen (¹⁵N). We combined the Electrical Penetration Graph (EPG) technique that allows detailed recording of aphid feeding behaviour and stable isotope ratio mass spectrometry (IRMS) to precisely measure the uptake of nitrogen. Bird cherry-oat aphids <i>Rhopalosiphum padi</i> L. (Hemiptera, Aphididae) fed for 24 h on barley plants (<i>Hordeum vulgare</i> L., cultivar Lina, Poaceae) that were cultivated with a ¹⁵N enriched nutrient solution. The time aphids fed in the phloem was strongly positive correlated with their ¹⁵N uptake. All other single behavioural phases were not correlated with ¹⁵N enrichment in the aphids, which corroborates their classification as non-feeding EPG phases. In addition, phloem-feeding and ¹⁵N enrichment of aphids was divided into two groups. One group spent only short time in the phloem phase and was unsuccessful in nitrogen acquisition, while the other group displayed longer phloem-feeding phases and was successful in nitrogen acquisition. This suggests that several factors such as the right feeding site, time span of feeding and individual conditions play a role for the aphids to acquire nutrients successfully. The power of this combination of methods for studying plant-aphid interactions is discussed.</p></div

Pearson correlation coefficient matrix.

<p>Significant correlation coefficients (p≤0.05) are written in bold numbers. Abbreviations: E2 = phloem feeding, E1 = pre-phloem, G = xylem feeding, C = pathway phase, F = derailed stylet mechanics, FI = feeding interruptions, non-feeding = sum of E1, G, C and F phase.</p