Journal Article

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

Frank Gaupels, Anja Buhtz, Torsten Knauer, Sachin Deshmukh, Frank Waller, Aart J. E. van Bel, Karl-Heinz Kogel and Julia Kehr

in Journal of Experimental Botany

Published on behalf of Society for Experimental Biology

Volume 59, issue 12, pages 3297-3306
Published in print September 2008 | ISSN: 0022-0957
Published online July 2008 | e-ISSN: 1460-2431 | DOI: http://dx.doi.org/10.1093/jxb/ern181

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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.

Keywords: Aphid; barley; cDNA-AFLP; mRNA; phloem; protein; Rhopalosiphum padi; signalling; stylectomy; two-dimensional gel electrophoresis

Journal Article.  5531 words.  Illustrated.

Subjects: Plant Sciences and Forestry

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