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JXB Advance Access originally published online on July 15, 2008
Journal of Experimental Botany 2008 59(12):3297-3306; doi:10.1093/jxb/ern181
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap

Frank Gaupels1,2,*, Anja Buhtz3, Torsten Knauer2, Sachin Deshmukh1, Frank Waller1, Aart J. E. van Bel2, Karl-Heinz Kogel1 and Julia Kehr3 {dagger}

1Institute of Phytopathology and Applied Zoology, IFZ, Heinrich-Buff-Ring 26–32, D-35392 Gießen, Germany
2Plant Cell Biology Research Group, Institute of General Botany, Senckenbergstrasse 17, D-35390 Gießen, Germany
3Max Planck Institute of Molecular Plant Physiology, Department Lothar Willmitzer, Am Mühlenberg 1, D-14424 Golm/Potsdam, Germany

* Present address and to whom correspondence should be addressed: Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada le Grazie 15, I-37134 Verona, Italy. E-mail: frank.gaupels{at}univr.it

Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 µl of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies.

Key words: Aphid, barley, cDNA-AFLP, mRNA, phloem, protein, Rhopalosiphum padi, signalling, stylectomy, two-dimensional gel electrophoresis

{dagger} Present address: Centro de Biotecnología y Genómica de Plantas (CBGP), Madrid, Spain

Received 21 April 2008; Revised 3 June 2008 Accepted 16 June 2008


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