Functional analysis of Brassica napus phloem protein and ribonucleoprotein complexes.

Phloem sap contains a large number of macromolecules, including proteins and RNAs from different classes. Proteome analyses of phloem samples from different plant species under denaturing conditions identified hundreds of proteins potentially involved in diverse processes. Surprisingly, these studies also found a significant number of ribosomal and proteasomal proteins. This led to the suggestion that active ribosome and proteasome complexes might be present in the phloem, challenging the paradigm that protein synthesis and turnover are absent from the enucleate sieve elements of angiosperms. However, the existence of such complexes has as yet not been demonstrated. In this study we used three-dimensional gel electrophoresis to separate several protein complexes from native phloem sap from Brassica napus. Matrix-assisted laser desorption ionization-time of flight MS analyses identified more than 100 proteins in the three major protein-containing complexes. All three complexes contained proteins belonging to different ribosomal fragments and blue native northern blot confirmed the existence of ribonucleoprotein complexes. In addition, one complex contained proteasome components and further functional analyses confirmed activity of a proteasomal degradation pathway and showed a large number of ubiquitinated phloem proteins. Our results suggest specialized roles for ubiquitin modification and proteasome-mediated degradation in the phloem.

Protocol: optimisation of a grafting protocol for oilseed rape (Brassica napus) for studying long-distance signalling.

BACKGROUND: Grafting is a well-established technique for studying long-distance transport and signalling processes in higher plants. While oilseed rape has been the subject of comprehensive analyses of xylem and phloem sap to identify macromolecules potentially involved in long-distance information transfer, there is currently no standardised grafting method for this species published. RESULTS: We developed a straightforward collar-free grafting protocol for Brassica napus plants with high reproducibility and success rates. Micrografting of seedlings was done on filter paper. Grafting success on different types of regeneration media was measured short-term after grafting and as the long-term survival rate (>14 days) of grafts after the transfer to hydroponic culture or soil. CONCLUSIONS: We compared different methods for grafting B. napus seedlings. Grafting on filter paper with removed cotyledons, a truncated hypocotyl and the addition of low levels of sucrose under long day conditions allowed the highest grafting success. A subsequent long-term hydroponic cultivation of merged grafts showed highest survival rates and best reproducibility.