Protein Profiles of Lipid Droplets during the Hypersensitive Defense Response of Arabidopsis against Pseudomonas Infection

Abstract

Lipid droplets (LDs) have classically been viewed as seed storage particles, yet they are now emerging as dynamic organelles associated with developmental and stress responses. Nevertheless, their involvement in plant immunity has still been little studied. Here, we found LD accumulation in Arabidopsis thaliana leaves that induced a hypersensitive response (HR) after Pseudomonas infection. We established a protocol to reproducibly isolate LDs and to analyze their protein content. The expression of GFP fusion proteins in Nicotiana benthamiana and in transgenic Arabidopsis lines validated the LD localization of glycerol-3-phosphate acyltransferase 4 (GPAT4) and 8 (GPAT8), required for cutin biosynthesis. Similarly, we showed LD localization of α-dioxygenase1 (α-DOX1) and caleosin3 (CLO3), involved in the synthesis of fatty acid derivatives, and that of phytoalexin-deficient 3 (PAD3), which is involved in camalexin synthesis. We found evidence suggesting the existence of different populations of LDs, with varying protein contents and distributions. GPAT4 and GPAT8 were associated with LDs inside stomata and surrounding cells of untreated leaves, yet they were mainly confined to LDs in guard cells after bacterial inoculation. By contrast, α-DOX1 and PAD3 were associated with LDs in the epidermal cells of HR-responding leaves, with PAD3 mostly restricted to cells near dead tissue, while CLO3 had a more ubiquitous distribution. As such, the nature of the proteins identified, together with the phenotypic examination of selected mutants, suggests that LDs participate in lipid changes and in the production and transport of defense components affecting the interaction of plants with invading pathogens.

Degradation of Lipid Droplets in Plants and Algae—Right Time, Many Paths, One Goal

“…2-HPOT is then converted by CLO3 into 2-hydroxy-octadecatrienoic acid (2-HOT), which possesses anti-fungal activity (Shimada et al, 2014). Additionally, accumulation of phytoalexin deficient 3 (PAD3) protein involved in camalexin synthesis was observed on the surface of leaf LDs after Pseudomonas syringae pv tomato (Pst) DC3000 avrRpm1 infection, supporting the role of leaf LDs in the plant defense response (Fernandez-Santos et al, 2020). Besides caleosin, three isoforms of Lipid Droplet Associated Proteins (LDAPs) have been identified in the leaves of Arabidopsis.…”

“…Depending on developmental stage and/or physiological state of the plant, LDs can also be present in non-seed organs such as fruits, leaves, stems, and roots (Lersten et al, 2006;Shimada et al, 2015;Pyc et al, 2017a;Huang, 2018). Leaf LDs seem to be equipped with a different set of integral proteins than seeds (Table S1) (Pyc et al, 2017a;Fernandez-Santos et al, 2020). A leaf-specific isoform of caleosin, CLO3, has been shown to decorate leaf LDs and to be directly involved in triggering the oxylipin-mediated response to pathogen attack in A. thaliana (Hanano et al, 2015;Shimada et al, 2015).…”

“…LDAPs have been shown to be essential for the maintenance and regulation of LD metabolism and to play nonredundant functions in the stress response and post-germinative growth ( Gidda et al., 2016 ). In addition to the above mentioned LD-associated proteins, a few proteins with diverse functions were also identified in the proteome of leaf LDs, including LDAP-interacting protein (LDIP) ( Pyc et al., 2017b ), glycerol-3-phosphate-acyltransferase 4 (GPAT4) and 8 (GPAT8) ( Fernandez-Santos et al., 2020 ), strictosidine synthase (STR), 2-oxoglutarate (2OG) and farnesylcysteine lyase (FCLY) ( Brocard et al., 2017 ). These observations suggest the multifunctional nature of LDs and support the direct involvement of non-seed LDs in stress-response and defense-related pathways in plants.…”

“…This indicates their highly conserved role in cellular lipid metabolism. Until recently LDs were considered as a simple TAG storage compartment, however, intense studies in the last decade revealed that LDs represent highly dynamic structures, involved in a plethora of diverse cellular processes, like regulation of energy homeostasis, remodeling of membranes, and signaling (Chitraju et al, 2017;Welte and Gould, 2017;Yang and Benning, 2018;Fernandez-Santos et al, 2020). According to the common structural model, LDs are composed of a hydrophobic core filled mostly with TAGs surrounded by a phospholipid monolayer, and are decorated with a set of specific proteins (Figure 1).…”

Co-option of a seed-like proteome by oil-rich tubers

“…Most plant cells store TAG predominantly in cytosolic lipid droplets (LDs) (31). LDs are presumably present in all cell types but can strongly differ in their protein composition (18,(32)(33)(34)(35). The described similarities between tubers and seeds and the notion that both accumulate oil raises the question if the tuber proteins that are associated with LDs are also similar to seeds.…”