de novo specification of a post-embryonic stem cell population in plants

植物从空气中吸收二氧化碳并将其转化为糖。大部分的糖被用于制造地球上最丰富的生物聚合物——纤维素。纤维素是木材的主要成分，木材是一种可再生的生物材料和生物能源的来源。该提案旨在阐明木质组织是如何形成的。木材是植物维管系统的一部分，由木质部细胞组成。木质部由一组被称为形成层的干细胞产生。了解形成层的功能可以用来增加林业环境中的木材形成，但在这些实验中，我们将使用模式植物，拟南芥，因为它很容易在实验室中操作。在植物和动物中，干细胞通常是在胚胎中形成的。形成层是不寻常的，因为它的干细胞群是在胚胎发生后形成的，这使它成为理解干细胞起始的一个有趣的系统。之前我们已经确定了六种信号蛋白，即所谓的受体激酶，它们共同促进形成层形成所必需的细胞分裂。然而，虽然我们知道它们在这个过程中很重要，但我们并不确切知道它们是如何工作的。在这项建议中，我们旨在回答这个问题。受体激酶定位于形成层细胞的质膜。一种假设是，蛋白质与蛋白质之间的相互作用发生在六种受体激酶中的某些激酶之间。我们将用两种方法来检验这个假设。首先，我们将用不同的荧光蛋白标记有问题的受体激酶。我们可以用显微镜确定两种不同的荧光蛋白之间是否发生了能量转移（称为FRET）。因为能量转移只有在两种蛋白质非常接近的情况下才有可能，所以我们只有在标记的受体激酶之间发生蛋白质-蛋白质相互作用时才会检测到FRET。我们将使用的第二种方法被称为共免疫沉淀。在这里，我们将使用一种抗体来靶向我们放置在受体激酶上的标签。如果受体激酶形成蛋白质复合体的一部分，我们将能够识别复合体的其他部分。受体激酶是一种向细胞膜上的细胞发送信号的分子。我们已经确定的六种受体激酶存在于几种细胞类型中，但是哪种细胞类型是正常形成层起始所必需的呢？这些蛋白能在几种细胞类型中启动形成层吗？我们的目标是在我们的建议中处理这个问题。我们已经产生了一个突变体，其中所有六种受体激酶都缺失了。我们建议在特定的细胞类型（包括木质部和形成层）以及其他维管细胞类型中替换这些蛋白质。这些实验将告诉我们受体激酶信号传导的结果是否局限于它们存在的细胞（称为细胞自主），或者它们是否影响邻近细胞的行为。最后，我们建议确定信号的结果是什么。受体激酶向细胞核发出信号以改变基因活性，但我们不知道哪些基因对受体激酶有反应，这是本研究的重点。我们建议通过受体激酶增加信号传导，并通过测序信号传导增加前后的mRNA来检测哪些基因有反应。mRNA水平的变化将告诉我们哪些基因受到了调控。它还将告诉我们是否某些受体激酶可以影响其他五种受体激酶的表达水平。总之，这些实验将告诉我们形成层是如何起作用的。他们还将提供关于如何在胚胎发生后建立干细胞群的信息。它们还可能为我们提供操纵木材形成以提高森林生产力和改善碳捕获的方法。

Plants draw carbon dioxide from the air and convert it to sugars. Much of the sugar is utilised in the most abundant biopolymer on Earth, cellulose. Cellulose is the main constituent of wood, and wood represents a renewable biomaterial and a source of bioenergy. This proposal aims to shed light on how woody tissue is formed. Wood is part of the plant vascular system and is made up of xylem cells. The xylem arises from a group of stem cells referred to as the cambium. Understanding of how the cambium functions could be used to increase wood formation in a forestry setting, but in these experiments we will use the model plant, Arabidopsis, because it is easy to manipulate in the lab.In both plants and animals, stem cells are typically formed in the embryo. The cambium is unusual because its stem cell population is formed after embryogenesis, making it an interesting system for understanding stem cell initiation. Previously we have identified six signalling proteins, so-called receptor kinases, which act together to promote the cell divisions necessary for cambium formation. However, while we know that they are important in this process, we don't know exactly how they work. In this proposal we aim to answer this question. The receptor kinases are localised to the plasma membrane in the cambium cells. One hypothesis is that protein-protein interactions occur between some of the six receptor kinases. We will test this hypothesis in two ways. In the first, we will tag the receptor kinases in question with different fluorescent proteins. We can determine if energy transfer between two different fluorescent proteins has occurred (referred to as FRET) using microscopy. Because energy transfer is only be possible if the two proteins are in very close proximity, we would only detect FRET if protein-protein interactions were occurring between the tagged receptor kinases. The second method that we will use is referred to as co-immunoprecipitation. Here we will use an antibody that targets a tag which we have placed on a receptor kinase. If the receptor kinase forms part of a protein complex we will be able to identify other parts of the complex.Receptor kinases are molecules that send signals to the cells on the membranes of which they are present. The six receptor kinases that we have identified are present on several cell types, but which cell type(s) are required for normal cambium initiation? Can these proteins initiate cambium in several cell types? We aim to address this question in our proposal. We have generated a mutant in which all six receptor kinases are missing. We propose to replace these proteins in specific cell types including the xylem and cambium, but other vascular cell types too. These experiments will tell us if the consequences of the receptor kinase signalling are restricted to the cell in which they are present (referred to as cell-autonomous), or if they influence the behaviour of neighbouring cells.Finally, we propose to determine what the outcome of the signalling is. Receptor kinases signal to the nucleus to alter gene activity, but we don't which genes respond to the receptor kinases that are the focus of this study. We propose to increase signalling through the receptor kinases and detect which genes respond by sequencing the mRNA before and after the signalling increase. Changes in mRNA levels will tell us which genes have been regulated. It will also tell us whether some of the receptor kinases can influence the levels at which the other five are expressed.Together, these experiments will tell us how the cambium functions. They will also provide information on how stem cell populations can be set up following embryogenesis. They may also suggest ways in which we can manipulate wood formation to increase forest productivity and improve carbon capture.