Evolution of lipid droplet-associated proteins and their role in drought resistance

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Drought is a prime stressor that plants had to overcome in their conquer of land. Most land plants are able to adapt to drought on the cellular level. Furthermore, they are able to produce desiccation tolerant cells and tissues as part of their reproductive cycle such as seeds, pollen and spores. However, many streptophyte algae are also displaying high stress resilience. While it is likely that drought resistance evolved several times independently, it is also likely that these independent origins of desiccation tolerance are underpinned by similar strategies and the co-option of existing regulatory programs for resilience. Common to both drought resistance and desiccation tolerance is the accumulation of neutral lipids, foremost triacylglycerol (TAG), in cytosolic lipid droplets (LDs) also referred to as oil bodies, lipid bodies or oleosomes. Several protein families are known to localize to LDs and two of them caleosin (CLO) and lipid droplet associated protein (LDAP) are associated with drought stress responses and possibly evolved this function in streptophyte algae. The goal of the research project is to find out if the accumulation of neutral lipids in LDs is a universal drought stress and desiccation response in the clade of Streptophyta by investigating the five species Mesotaenium endlicherianum, Zygnema circumcarinatum, Physcomitrium patens, Marchantia polymorpha and Azolla filiculoides. Furthermore, we want to collect evidence that LD-associated proteins that are upregulated during drought are important to cope with this stress, and evolved this function already in streptophyte algae. Overall, this data would support the hypothesis that the accumulation of LDs and its associated proteins during drought was one of the prerequisites for the conquer of land. The key objectives are to collect evidence for the following hypotheses: 1. The remodeling of the lipidome and the accumulation of neutral lipids in LDs is a universal stress response. We will monitor the abundance of LDs by microcopy and perform lipidome analyses in drought-treated vegetative tissues of the five species. 2. The proteins CLO and LDAP are universally upregulated under desiccation and drought stress, respectively. We will test the transcript of all respective genes by qPCR in all five species and isolate LDs from drought stressed tissues to analyze these by proteomics. 3. CLO and LDAP are important during drought stress and/or in spores in P. patens. We will knock out the three CLO and the single LDAP gene by CRISPR/Cas9 and analyze the phenotypes in respect to LD number and size, drought resistance and the performance of the spores. 4. CLO and LDAP have kept a similar function throughout evolution from streptophyte algae to land plants. We will complement the P. patens mutant lines that have displayed phenotypes with homolog genes from M. endlicherianum.