Title: Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three proteins are indeed lipid-binding and act in the vasculature possibly in a function related to long-distance signaling, the three proteins do not act in the same but rather in distinct pathways. Furthermore, it points toward PLAFP as a prime candidate to investigate long-distance lipid signaling in the plant drought response.