Identification and Characterisation of Lipid Droplet-Localised Proteins

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Lipid droplets (LD) are unique organelles, whose physiological relevance has been underestimated for a long time. Well-known in seed tissues for storing triacylglycerol (TAG) to fuel post-germinative seedling establishment, LDs in plants are nowadays known to be involved in many more processes. They also present a highly dynamic hub for cellular lipids through the short-term turnover of lipids. They are involved in lipid homeostasis and remodelling of various cellular membranes, e.g. through regulating the balance of lipid storage versus membrane expansion or by storing and buffering excess, unneeded or toxic lipids. This sink – rather than source – function of LDs is especially important during various stresses, such as e.g. heat stress. Here, membranes quickly have to adapt their membrane properties to maintain membrane integrity and prevent hyperfluidity, i.e. unsaturated fatty acids (FAs) with low melting points have to be replaced by saturated FAs with higher melting points. Indeed, LDs are observed to accumulate in response to different abiotic stresses, including heat stress, and with their lipid buffering functions they are often involved in amelioration of the stress. To fulfil their roles in maintaining lipid homeostasis of various organellar membranes, especially during stress conditions, contacts of LDs with these organelles are important. These membrane contact sites (MCS) of LDs are well-described in yeast and mammals and likewise often increase in response to stress. In mammals and yeast, they have been observed between LDs and nearly all other organelles. Data on plant LD contact sites, however, is scarce and limited to LD-ER and LD-peroxisome contacts. In the present study, I shed light on the involvement of LD-stored TAG in the process of Nicotiana tabacum pollen tube heat adaptation. Pollen tubes are reproductive tissues highly sensitive to abiotic stresses and rich in LDs. Proper growth of the pollen tube is crucial for fertilisation and pollen competition and the ability to quickly react to changing environments is of upmost importance. We here show that heat-stress causes quick membrane lipid remodelling in ex vivo grown pollen tubes, leading to a pronounced decrease of unsaturated fatty acids (FAs) in nearly all phospholipid classes. Concomitantly, an unspecific increase in TAG and a significant increase in transcripts of several Tobacco DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) isoforms is observed. We propose a model, where heat stress causes unspecific bulk membrane lipid degradation in growing pollen tubes, and released FAs are channelled into TAGs in a DGAT1-dependant way to be replaced in the membranes by newly synthesised saturated FAs, thereby ensuring correct membrane fluidity. Furthermore, I identify a putative MCS between LDs and the plasma membrane (PM) in Arabidopsis thaliana seeds and seedlings. Two formerly undescribed proteins, SEED LIPID DROPLET PROTEIN (SLDP) 1 and 2, were shown to be LD-localised proteins. Mutations of these proteins cause aberrant clustering of LDs that normally evenly distribute along the PM during germination in Arabidopsis thaliana. Mutation of a putative interaction partner, PM-localised LIPID DROPLET PM ANCHOR (LIPA), was found to cause the same phenotype. In addition, a mutual recruitment of ectopically expressed LIPA and SLDP to the PM and LDs, respectively, was observed in pollen tubes. By expressing both proteins in this non-native tissue, a seedling-like distribution of LDs could be reconstituted: LDs, that usually float dynamically through the cytoplasm in pollen tubes are immobilised along the PM when both proteins are co-expressed. Taken together, we suggest a model, where proper cellular LD distribution along the PM in germinating A. thaliana is achieved through a MCS between LD-localised SLDP and PM-localised LIPA.