Analysis of regulatory networks underlying morphogenesis of leaf epidermis pavement cells

从受精卵细胞到具有特殊组织和器官的复杂生物体的多细胞生物的发育需要精确的生长协调。驱动生长的基本过程是细胞分裂和细胞扩增，它们分别产生新细胞和增加单个细胞的大小。在细胞扩增过程中，细胞获得各种不同的形状以实现其特殊功能（细胞分化）。在植物中，细胞的增殖受到细胞壁的控制和限制，细胞壁在质膜外包围着单个细胞，并物理地粘合邻近的细胞。细胞壁物质的沉积是由植物细胞骨架介导的，细胞骨架由微管和肌动蛋白丝组成，形成一个高度动态的细胞内网络。细胞壁和细胞骨架的协同活动是由复杂的信号网络协调的，这些信号网络整合了内源性和外源性信号，以精确调节生长。叶表皮铺装细胞是植物中最复杂的细胞形态之一，具有多个裂片和背斜壁上的凹痕。除了对叶片生长的重要性和对光合活性组织的保护作用外，路面细胞因此是在机械偶联细胞组织中研究多极生长的一个流行和合适的模型系统。遗传和药物研究，主要是在模式植物拟南芥中，已经确定了控制路面细胞形态发生的几个因素。然而，形状形成的精确机制和原理在很大程度上仍然是个谜。在我们之前的工作中，我们发现了植物特异性微管相关蛋白类的成员IQ67 DOMAIN5 (IQD5)，作为路面细胞形状和细胞壁组成的新调节剂，可能在微管细胞骨架上整合钙信号。此外，我们还开发了一种软件工具，首次实现了从显微镜图像中对路面细胞形状的全自动检测和比较定量分析。我们对自然发生的拟南芥生态型路面细胞形状的首次分析揭示了大的种内变异，为鉴定相关遗传位点提供了合适的基础。在拟议的项目中，我们的目标是利用细胞生物学和反向遗传学方法，从功能上表征IQD5在调节微管组织和细胞信号通路中的作用。此外，我们将使用前向遗传学方法，通过将拟南芥的基因型和表型变异联系起来，并通过筛选突变体来鉴定新的调节因子。总的来说，通过反向和正向遗传学方法对路面细胞形状调节的综合分析将为研究细胞形态发生机制提供一个合适的框架，并将有助于对潜在原理的更全面的理解。

The development of multicellular organisms from a fertilized egg cell to complex organisms with specialized tissues and organs requires precise coordination of growth. The basic processes that drive growth are cell division and cell expansion, which generate new cells and increase the size of individual cells, respectively. During cell expansion, cells acquire various distinct shapes to fulfill their specialized functions (cell differentiation). In plants, cell expansion is controlled and restricted by a rigid cell wall that surrounds individual cells outside of the plasma membrane and physically glues neighboring cells. Deposition of cell wall material is mediated by the plant cytoskeleton, comprised of microtubules and actin filaments, which form a highly dynamic intracellular network. The concerted activity of the cell wall and the cytoskeleton is coordinated by complex signaling networks that integrate endogenous and external signals for precise regulation of growth. Leaf epidermis pavement cells form one of the most complex cell shapes in plants with multiple lobes and indentations in their anticlinal walls. In addition to their importance for leaf growth and their protective role for photosynthetically active tissues, pavement cells thus are a popular and suitable model system to study multipolar growth in the context of a tissue of mechanically coupled cells. Genetic and pharmaceutical studies, mostly in the model plant Arabidopsis thaliana, have identified several factors that control pavement cell morphogenesis. The precise mechanisms and principles underlying shape formation, however, remain largely enigmatic. In our previous work we identified a member of a plant-specific class of microtubule-associated proteins, IQ67 DOMAIN5 (IQD5), as novel regulator of pavement cell shape and cell wall composition, which likely integrates calcium signaling at the microtubule cytoskeleton. In addition, we have developed a software-tool, which for the first time enables fully automatic detection and comparative quantitative analysis of pavement cell shape from microscopy images. Our first analyses of pavement cell shape in naturally occurring Arabidopsis ecotypes revealed large intraspecific variation that provide a suitable basis to identify associated genetic loci. Within the proposed project we aim to functionally characterize the role of IQD5 in regulation of microtubule organization and in cellular signaling pathways, using cell biology and reverse-genetics approaches. In addition, we will use forward-genetics approaches to identify novel regulators by linking genotypic and phenotypic variation within Arabidopsis accessions and by screening of mutant collections. Collectively, the combined analysis of pavement cell shape regulation by reverse- and forward-genetics approaches will provide a suitable framework to investigate mechanisms of cell morphogenesis and will contribute to a more holistic understanding of the underlying principles.