Identification and characterization of phloem and xylem sap proteins in Populus trichocarpa x P. deltoides




Dafoe, Nicole

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Hundreds of proteins have been detected in phloem and xylem sap, even though the cells involved in long distance phloem and xylem transport are incapable of protein synthesis at maturity. We are now beginning to learn the identity and function of these proteins, but this knowledge is generally limited to annual plants. The first objective of this study was to identify phloem and xylem sap proteins in the perennial, poplar. Using LC-MS/MS, 48 proteins were identified in poplar phloem exudate and 98 proteins were identified in xylem sap. A large number of phloem exudate proteins are insect defense proteins that include protease inhibitors and polyphenol oxidase and also members of the pop3/SPI gene family, whereas a number of xylem sap proteins were pathogenesis-related proteins such as thaumatin-like proteins (TLPs) and chitinases that function in pathogen defense. The importance of xylem sap proteins in pathogen defense has been previously demonstrated, however, the role of phloem proteins in insect defense is currently unknown. A major question to be addressed in order to understand the function of phloem proteins is if they are differentially regulated in response to insect herbivory. The second objective of this study was to identify poplar phloem proteins differentially regulated in response to simulated insect feeding. Using two-dimensional gel electrophoresis, two proteins, PtTLP1 and pop3.1, were consistently upregulated 24 hours post-wounding. The third objective of this study was to produce antibodies against these proteins to use to further characterize their expression and localization patterns. Antibodies were also produced against another phloem exudate protein, pop3.4, which is 40% similar to pop3.1. The origin of all three proteins inside sieve elements was confirmed with immunolocalization. PtTLP 1, pop3.1 and pop3.4 antisera labelled organelle-like structures in sieve elements and also phloem parenchyma cells. For PtTLP1, these structures were identified as starch and starch containing plastids. All three antisera also labelled cell wall proteins in different cell types. Overall, this study represents the first large-scale analysis of phloem and xylem sap proteomes from a perennial and describes the first observation of wound-inducible phloem sap proteins.



Poplar, Phloem, Xylem