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

干旱是植物在征服土地的过程中必须克服的主要压力。大多数陆地植物能够在细胞水平上适应干旱。此外，它们能够产生耐脱水细胞和组织，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强的抗逆性。虽然抗旱性可能独立进化了几次，但这些干燥耐受性的独立起源也可能是由类似的策略和现有监管计划的共同选择来支撑的。抗旱性和耐干性的共同之处是中性脂类的积累，最重要的是三酰甘油(TAG)在细胞质脂滴(LDS)中积累，也称为油体、脂体或油体。已知有几个蛋白家族定位于LDS，其中两个与干旱胁迫反应有关的蛋白家族是钙黄蛋白(CLO)和脂滴相关蛋白(LDAP)，并可能在链藻中进化出这一功能。本研究的目的是通过对5种植物的研究，确定中性脂类物质的积累是否是链霉属的一种普遍的干旱胁迫和干燥反应。此外，我们希望收集证据表明，在干旱期间上调的LD相关蛋白对于应对这种胁迫是重要的，并且已经在链藻中进化出了这种功能。总体而言，这些数据将支持这样一种假设，即干旱期间LDS及其相关蛋白质的积累是征服土地的先决条件之一。研究的主要目的是为以下假说收集证据：1.腰椎管结节的脂膜重塑和中性脂类的积聚是一种普遍的应激反应。我们将通过显微镜监测LDS的丰度，并在干旱处理的五个物种的营养组织中进行脂组分析。2.干旱胁迫和干旱胁迫分别普遍上调了CLO和LDAP蛋白的表达。我们将通过定量聚合酶链式反应检测所有五个物种各自基因的转录本，并从干旱胁迫组织中分离LDS，以进行蛋白质组学分析。3.在干旱胁迫过程中和/或在白粉菌孢子中，CLO和LDAP是重要的。我们利用CRISPR/Cas9敲除了3个ClO基因和1个ldap基因，并从LD数量和大小、抗旱性和孢子的性能等方面对其表型进行了分析。4.在从链藻到陆生植物的整个进化过程中，CLO和LDAP一直保持着相似的功能。我们将补充P.patens突变株，这些突变株的表型与根结线虫的同源基因相同。

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.