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

干旱是植物征服土地时必须克服的主要压力源。大多数陆地植物都能在细胞水平上适应干旱。此外，它们能够产生耐干燥的细胞和组织，作为其生殖周期的一部分，如种子、花粉和孢子。然而，许多链藻也表现出很高的应激恢复能力。虽然抗旱性很可能是独立进化了几次，但也有可能这些抗旱性的独立起源是由类似的策略和现有的适应性调节计划共同支持的。抗旱性和耐干燥性的共同特点是中性脂质的积累，主要是三酰甘油（TAG），在细胞质脂滴（ld）中也被称为油体、脂体或油小体。已知有几个蛋白家族定位于lld，其中两个与干旱胁迫反应有关的蛋白家族是caleosin （CLO）和脂滴相关蛋白（LDAP），它们可能在链藻中进化出了这种功能。本研究目的是通过对中链菌（Mesotaenium endlicherianum）、环扎菌（Zygnema circumcarinatum）、白质菌（Physcomitrium patens）、多形药（Marchantia polymorpha）和杜鹃花（Azolla filiculoides） 5种植物的研究，探讨中性脂的积累是否为链菌纲普遍存在的干旱胁迫和干燥反应。此外，我们希望收集证据，证明干旱期间上调的ld相关蛋白对应对这种压力很重要，并且已经在链藻中进化出了这种功能。总的来说，这些数据将支持这样的假设，即干旱期间ld及其相关蛋白质的积累是征服土地的先决条件之一。主要目标是为以下假设收集证据：脂质体的重塑和中性脂质的积累是一种普遍的应激反应。我们将通过显微复制监测ld的丰度，并对干旱处理过的五种植物的营养组织进行脂质组分析。2. CLO和LDAP蛋白在干燥和干旱胁迫下普遍上调。我们将通过qPCR检测所有五个物种中所有相关基因的转录本，并从干旱胁迫组织中分离ld，通过蛋白质组学分析这些基因。3. CLO和LDAP在干旱胁迫和/或孢子中起重要作用。我们将利用CRISPR/Cas9基因敲除三个CLO基因和一个LDAP基因，并分析其在LD数量和大小、抗旱性以及孢子性能方面的表型。4. CLO和LDAP在从链藻到陆生植物的进化过程中一直保持着相似的功能。我们将用同源基因从M. endlicherianum补充已经显示表型的P. patens突变系。

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.