Proteomic Analysis of Lipid Droplets from Arabidopsis Aging Leaves Brings New Insight into Their Biogenesis and Functions

Abstract

Lipid droplets (LDs) are cell compartments specialized for oil storage. Although their role and biogenesis are relatively well documented in seeds, little is known about their composition, structure and function in senescing leaves where they also accumulate. Here, we used a label free quantitative mass spectrometry approach to define the LD proteome of aging Arabidopsis leaves. We found that its composition is highly different from that of seed/cotyledon and identified 28 proteins including 9 enzymes of the secondary metabolism pathways involved in plant defense response. With the exception of the TRIGALACTOSYLDIACYLGLYCEROL2 protein, we did not identify enzymes implicated in lipid metabolism, suggesting that growth of leaf LDs does not occur by local lipid synthesis but rather through contact sites with the endoplasmic reticulum (ER) or other membranes. The two most abundant proteins of the leaf LDs are the CALEOSIN3 and the SMALL RUBBER PARTICLE1 (AtSRP1); both proteins have structural functions and participate in plant response to stress. CALEOSIN3 and AtSRP1 are part of larger protein families, yet no other members were enriched in the LD proteome suggesting a specific role of both proteins in aging leaves. We thus examined the function of AtSRP1 at this developmental stage and found that AtSRP1 modulates the expression of CALEOSIN3 in aging leaves. Furthermore, AtSRP1 overexpression induces the accumulation of triacylglycerol with an unusual composition compared to wild-type. We demonstrate that, although AtSRP1 expression is naturally increased in wild type senescing leaves, its overexpression in senescent transgenic lines induces an over-accumulation of LDs organized in clusters at restricted sites of the ER. Conversely, atsrp1 knock-down mutants displayed fewer but larger LDs. Together our results reveal that the abundancy of AtSRP1 regulates the neo-formation of LDs during senescence. Using electron tomography, we further provide evidence that LDs in leaves share tenuous physical continuity as well as numerous contact sites with the ER membrane. Thus, our data suggest that leaf LDs are functionally distinct from seed LDs and that their biogenesis is strictly controlled by AtSRP1 at restricted sites of the ER.

Arabidopsis lipid droplet‐associated protein (LDAP) – interacting protein (LDIP) influences lipid droplet size and neutral lipid homeostasis in both leaves and seeds

“…LDIP was subsequently confirmed as a bona fide constituent of LDs using a variety of approaches, including co‐localizations with oleosin or LDAP at LDs in tobacco leaves (Figure ), isolation of LDs from germinating seedlings followed by Western blotting or proteomics, which showed clear enrichment of Arabidopsis LDIP in the LD fraction (Figure ), and stable expression of Cherry‐tagged LDIP in transgenic plants, which confirmed localization to LDs in Arabidopsis leaves and complemented the LDIP KO phenotype (Figure S10). Consistent with these data, LDIP was also identified as a constituent of LDs in a recent proteomics study of LDs isolated from Arabidopsis senescing leaves (Brocard et al ., ).…”

“…However, LDs are present in virtually all plant cell types, many of which are devoid of oleosins. This observation has recently led to increased efforts to identify LD proteins in non‐seed tissues/organs (Gidda et al ., ; Horn et al ., ; Davidi et al ., ; Huang and Huang, ; Brocard et al ., ), as well as to probe the function of LDs in cell types that do not specialize in fat storage (Shimada et al ., ; Gidda et al ., ; McLachlan et al ., ; Zhang et al ., ).…”

“…Further, despite similarities of the LDAPs in terms of their ability to modulate LD abundance, exposure of plants to heat or cold stress results in the differential induction of LDAP genes, and analysis of loss‐of‐function mutants confirmed that specific LDAP s are required for the proliferation of LDs under different stress conditions (Gidda et al ., ). Consistent with these results, the LDAPs have also been implicated in drought stress response as well as in overall plant growth and development (Kim et al ., , ; Seo et al ., ; Brocard et al ., ), suggesting broader roles for LDAPs and LDs in plant physiological processes and stress adaptations.…”

Membrane Dynamics and Multiple Functions of Oil Bodies in Seeds and Leaves

“…These combined results suggest that caleosins and their products function in stress response pathways. Proteomics analyses of leaf oil bodies detect abundant levels of CLO3 and a-DOX1 but not CLO4 (Shimada et al, 2014;Brocard et al, 2017), suggesting that leaf oil bodies containing CLO3 and a-DOX1 differ from those containing CLO4. Leaf oil bodies containing CLO3 and a-DOX1 are induced in senescent leaves (Shimada et al, 2014), whereas the developmental pattern of leaf oil bodies containing CLO4 is unknown.…”

“…Proteomic analyses of leaf oil bodies in senescent Arabidopsis leaves indicate that LDAP1 is localized on oil bodies ( Fig. 5A; Brocard et al, 2017). LDAP overexpression increases the abundance of dark-induced leaf oil bodies, whereas LDAP deficiency reduces their abundance (Gidda et al, 2016).…”