Analysis of the Pumpkin Phloem Proteome Provides Insights into Angiosperm Sieve Tube Function

Increasing evidence suggests that proteins present in the angiosperm sieve tube system play an important role in the long distance signaling system of plants. To identify the nature of these putatively non-cell-autonomous proteins, we adopted a large scale proteomics approach to analyze pumpkin phloem exudates. Phloem proteins were fractionated by fast protein liquid chromatography using both anion and cation exchange columns and then either in-solution or in-gel digested following further separation by SDS-PAGE. A total of 345 LC-MS/MS data sets were analyzed using a combination of Mascot and X!Tandem against the NCBI non-redundant green plant database and an extensive Cucurbit maxima expressed sequence tag database. In this analysis, 1,209 different consensi were obtained of which 1,121 could be annotated from GenBank and BLAST search analyses against three plant species, Arabidopsis thaliana, rice (Oryza sativa), and poplar (Populus trichocarpa). Gene ontology (GO) enrichment analyses identified sets of phloem proteins that function in RNA binding, mRNA translation, ubiquitinmediated proteolysis, and macromolecular and vesicle trafficking. Our findings indicate that protein synthesis and turnover, processes that were thought to be absent in enucleate sieve elements, likely occur within the angiosperm phloem translocation stream. In addition, our GO analysis identified a set of phloem proteins that are associated with the GO term “embryonic development ending in seed dormancy”; this finding raises the intriguing question as to whether the phloem may exert some level of control over seed development. The universal significance of the phloem proteome was highlighted by conservation of the phloem proteome in species as diverse as monocots (rice), eudicots (Arabidopsis and pumpkin), and trees (poplar). These results are discussed from the perspective of the role played by the phloem proteome as an integral component of the whole plant communication system. Molecular & Cellular Proteomics 8:343–356, 2009. In the plant kingdom, the vascular system is composed of xylem, which serves to deliver water and mineral nutrients from the soil to the aerial regions of the plant, and phloem, which delivers sugars and amino acids to developing organs. In the phloem system of flowering plants, the delivery of nutrients occurs through a highly specialized sieve tube system composed of files of nucleate companion cells that support the operation of their enucleate sieve elements (1). Recent studies have shown that the phloem translocation stream also serves as a conduit for the long distance delivery of proteins as well as mRNA, small RNA, and viral nucleic acids (2–16). These studies suggested that such phloem-mobile macromolecules may well participate in and coordinate developmental and physiological events at the whole plant level (17–20). Using grafting techniques, the translocation of specific RNA molecules and proteins from the stock into the recipient scion tissues was shown to correlate with changes in developmental and physiological phenotypes (8–12). A wide variety of proteins are also transported into and selectively delivered by the phloem translocation stream (4, 7, 20–31). For example, FLOWERING LOCUS T (FT), identified as a component of the florigenic signal in the angiosperms, is translocated through the phloem to the shoot apex where it induces flowering (11–15). Other studies have established that certain proteins involved in stress, defense, and an antioxidant defense system are also present within the phloem translocation stream (16, 29, 32). However, current knowledge on the process(es) and regulation of macromolecular trafficking within the phloem as well as the capacity of these signaling molecules to act within the context of a whole plant signaling network remains limited. A proteomics approach has been used to identify phloem proteins from several plant species. To date, proteomics anal-