Amino acid analysis by micellar electrokinetic chromatography with laser‐induced fluorescence detection: Application to nanolitre‐volume biological samples from Arabidopsis thaliana and Myzus persicae

Abstract

Amino acids were derivatised with 4-fluoro-7-nitrobenzo-2,1,3-oxadiazol (NBD-F), separated by micellar electrokinetic chromatography (MEKC), and detected by argon-ion (488 nm) laser-induced fluorescence. The optimised MEKC background electrolyte conditions were: 40 mM sodium cholate, 5 mM beta-cyclodextrin in 20 mM aqueous borate buffer, pH 9.1, with 7% v/v acetonitrile. Using these conditions, 19 amino acids were separated within 17 min. The limits of detection were in the range of 7.6-42.2 pmol/mL and limits of quantitation from 0.05-0.14 nmol/mL. The method was systematically validated for injection volume error, migration time variation, calibration linearity, accuracy, precision, and recovery. Nanolitre volume samples of phloem sap of individual sieve element cells from the plant Arabidopsis thaliana and honeydew from the aphid Myzus persicae were directly analysed with this method. Quantitative amino acid concentrations in these two biological matrices were profiled for the first time. This method is particularly important because it allows the complete profile of the amino acids obtained from individual phloem elements, allowing cell to cell and plant to plant variation to be quantified, which to date has not been possible with Arabidopsis thaliana.

Biosynthesis and Defensive Function of N Δ-Acetylornithine, a Jasmonate-Induced Arabidopsis Metabolite

“…This concentration of N d -acetylornithine was higher than what we observed in assays of whole leaves and other Arabidopsis tissue ( Figures 1B to 1F). MeJA elicitation in this experiment also caused a 24% decrease in exudation of the main phloem transport amino acids (Asn, Asp, Gln, and Glu; P < 0.01, t test; see Supplemental Figure 7 online), which together constitute ;70% of the total free amino acids in Arabidopsis phloem sap (Zhu et al, 2005). (A) N d -acetylornithine accumulation in wild-type Col-0 4 d after treatment with coronatine, coronatine-producing and nonproducing (Cor-) P. syringae strains Psm4326 and DC3000, and DC3000 carrying the avirulence genes avrRpt2 and avrRpm1.…”

“…N dacetylornithine was also abundant in phloem exudates collected from MeJA-treated Arabidopsis rosettes ( Figure 5B), where it constituted 12% of the free amino acids released from cut stems. Since aphid stylectomy experiments showed 49 mM total amino acid content in Arabidopsis phloem (Zhu et al, 2005), we estimated that there is 5.9 mM N d -acetylornithine in the phloem sap after MeJA elicitation. This concentration of N d -acetylornithine was higher than what we observed in assays of whole leaves and other Arabidopsis tissue ( Figures 1B to 1F).…”

A Diurnal Component to the Variation in Sieve Tube Amino Acid Content in Wheat

“…Air-dried samples were analyzed for amino acid content by CE-LIF based on the method of Zhu et al (2005). Briefly, the samples were derivatized with Figure 5.…”

“…Aphids feed exclusively from the phloem (for review, see Will and van Bel, 2006), and a large literature shows that their stylet bundles penetrate and extract phloem sap from an individual ST (Kimmins and Tjallingii, 1985;Tjallingii and Esch, 1993). It is possible to sever the stylets of feeding aphids and collect samples of phloem sap (Unwin, 1978;Pritchard, 1996;Zhu et al, 2005), and studies have confirmed that the samples obtained represent pure ST sap (Tjallingii and Esch, 1993). This technique has been used in previous studies to measure ST composition, and in particular amino acids, using HPLC for analysis.…”

“…We recently reported (Hunt et al, 2006) refinements of analytical techniques to measure amino acids (Zhu et al, 2005) in sap from individual exuding stylets using capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIF). CE-LIF is a powerful tool in bioanalysis, especially where sample volume is limited and high sensitivity is required (Tseng et al, 2007).…”