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

Abstract

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 TM 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.

Binding Properties of the N-Acetylglucosamine and High-Mannose N-Glycan PP2-A1 Phloem Lectin in Arabidopsis

“…We also cannot rule out the possibility that the method used to collect phloem sap, involving the use of EDTA, a chelator of Ca 2+ ions, affects the aggregation and mobility of PP2 proteins. Previous studies identifying PP2 phloem lectins in phloem sap proteomes were based on the collection of sap by phloem bleeding rather than EDTA-facilitated exudation (Walz et al, 2002(Walz et al, , 2004Giavalisco et al, 2006;Lin et al, 2009). Therefore, it remains unclear whether PP2-A1 subunits or small oligomers are mobile in the sieve elements of Arabidopsis or whether they are attached to sieve element membranes or to P-proteins.…”

“…Several putative LRRrelated proteins were also found. Two LRR proteins were found in C. maxima phloem sap (Lin et al, 2009). LRR domains are present in proteins with diverse functions, providing a framework for the formation of protein-protein interactions.…”

Arabidopsis NPCC6/NaKR1 Is a Phloem Mobile Metal Binding Protein Necessary for Phloem Function and Root Meristem Maintenance

“…This indicates that metal binding is important for NaKR1 function. HMA domain proteins have been identified in the phloem sap of Brassica napus and Cucurbita maxima (Giavalisco et al, 2006;Lin et al, 2009). In Arabidopsis, HMA domain protein CCH is found in the phloem and is thought to transport Cu out of senescing tissues (Himelblau et al, 1998;Mira et al, 2001).…”

Looking Deep Inside: Detection of Low-Abundance Proteins in Leaf Extracts of Arabidopsis and Phloem Exudates of Pumpkin

“…We used 1,080 mL of ProteoMiner-treated samples and 27 mL of control phloem samples and analyzed the extracts in 36 MS runs. Thus, in comparison with Lin et al (2009), our protocol for phloem proteomics afforded less than 4% of starting material, 10% of MS runs, and was overall more time and cost effective. While Lin et al (2009) employed elaborate methods for an exploratory genome study, the procedure presented here is compatible with high-throughput approaches.…”

“…The EFP is a unique feature of the Cucurbitaceae (Turgeon and Oparka, 2010;Atkins et al, 2011). In contrast to the fascicular phloem, the EFP does not build effective callose plugs upon wounding, which allows for the sampling of large volumes (50-200 mL plant 21 ) of spontaneously exuding phloem sap from cut stems and petioles (Van Bel and Gaupels, 2004;Lin et al, 2009;Turgeon and Oparka, 2010). Sugar concentrations in pumpkin phloem exudates are low, implying that the EFP of cucurbits does not function in assimilate transport but rather might be involved in defense and (systemic) signaling (Walz et al, 2004;Zhang et al, 2010).…”