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The correlation between leaf-surface and leaf-tissue secondary metabolites: a case study with pyrrolizidine alkaloids in Jacobaea hybrid plants

ABSTRACT: Introduction: Usually whole plant or whole leaf extracts are analyzed to study the chemical ecology of insect-plant interactions. For herbivore species the contact with the leaf surface enables them to estimate the quality of the plant. The relationship between the leaf-surface and leaf-tissue secondary metabolites (SMs) could offer important new insights in insect-plant interactions mediated by SMs. Pyrrolizidine alkaloids (PAs), typical defense chemicals in Jacobaea species, are repellent for generalist herbivores but are attractive to specialists.
Objectives: Explore whether the PAs on the leaf surface are a reliable representation of the PAs in the leaf tissue in PA-containing plants.
Method: The concentration of individual PAs present on the leaf surface and in the corresponding leaf tissue from 37 genotypes (one plant from each genotype) of an F2 generation of a cross between Jacobaea vulgaris and Jacobaea aquatica was measured by high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). PAs were removed from the leaf surface by extraction with a slightly acidic aqueous solution.
Results: The total amount of PAs present on the surface of the leaves was only 0.015% (range: 0.001-0.163%) of the total amount present in the leaf tissue. Most PAs present in the leaf tissue were also found on the surface, except for jaconine, dehydrojaconine, dehydrojacoline and usaramine N-oxide. Positive correlations between leaf-surface and leaf-tissue concentrations were found for most of the jacobine-like and otosenine-like PAs, but correlations for total PA, senecionine- and erucifoline-like PAs were not significant.
Conclusion: These results indicate that PA variation on the leaf surface only partially reflects the PA variation in the leaf tissue. Because most herbivores are affected in a different manner by individual PAs, this result means that the leaf surface does not give a reliable estimate of plant quality to herbivores.

INSTRUMENT(S): Quattro Premier XE (Waters)

SUBMITTER: Patrick Mulder

PROVIDER: MTBLS429 | MetaboLights | 2018-05-01

REPOSITORIES: MetaboLights

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			Action	DRS
	MTBLS429	Other
	FILES	Other
	a_MTBLS429_pas_leaf_surface_metabolite_profiling_mass_spectrometry.txt	Txt
	i_Investigation.txt	Txt
	m_MTBLS429_pas_leaf_surface_metabolite_profiling_mass_spectrometry_v2_maf.tsv	Tabular

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Project description:The investigation contains two sets of experiment. Set I. Transcriptional profiling of salt treated WT A. thaliana (WS ecotype) (100mM NaCl WT/0mM NaCl WT): Set II. Transcriptional profiling of salt treated ABR17- A. thaliana lines (100mM NaCl ABR17/0mM NaCl ABR17). Tissue for microarray analysis was obtained by placing surface sterilized seeds of A. thaliana (line 6.9) and the WT on half strength Murashige & Skoog (MS)(Murashige and Skoog, 1962) medium (1.5% sucrose, 0.8% agar with pH 5.7) medium in Petri dishes with or without 100mM NaCl at RT (21 ± 2 °C) after surface sterilization. The seeds were surface sterilized by rinsing with 70% ethanol for one minute, incubation in 20% bleach for 15 min and subsequent washes (four times for 5min each) to remove the bleach. MS plates with the seeds were placed at RT (21 ± 2 °C) under continuous fluorescent light 30 ?mol m-2 s-1 for 14 days. Seedlings (14 day-old) from three independently grown biological replicates in two set of experiments were removed from the MS plates, flash frozen in liquid nitrogen and stored at 80 °C until used for RNA extraction. Technical protocols for preparing the hybridization extract: 1. RNA was isolated using the QIAGEN RNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON, Canada) from two-week-old WT (grown on 0 and 100mMNaCl) and ABR17 (grown on 0 and 100mMNaCl) seedling tissue grown at three independent times (biological replicates). The integrity of all RNA samples assessed by agarose gel (1.2 percent) electrophoresis. 2. 6 ?g (Six micrograms) of total RNA was used to synthesize cDNAs using SuperScript® II RT (Invitrogen Inc., Burlington, ON, Canada) with RT polyA-capture primers in 3D Array 900TM (Genisphere Inc., Hatfield, PA, USA). Each pair of treated (100mM NaCl) and untreated (0mM NaCl) samples within each of the three biological replicates from two sets of experiments (100mM NaCl WT/0mM NaCl WT ; 100mM NaCl ABR17/0mM NaCl ABR17) was labelled in a reciprocal dye-swap design, for a total of 12 hybridizations. Note: In the transgenic plant production, the pea cDNA encoding for ABR17 was constitutively expressed under the control of Cauliflower mosaic virus 35S promoter. Reference: Srivastava S., Rahman H., Shah S. and Kav N.N.V. Constitutive expression of pea ABA-responsive 17 (ABR17) cDNA confers multiple stress tolerance in Arabidopsis thaliana. Plant Biotechnology Journal (2006) 4, 529-549.

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The correlation between leaf-surface and leaf-tissue secondary metabolites: a case study with pyrrolizidine alkaloids in Jacobaea hybrid plants

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