Functional analysis of the FORKED and FORKED-LIKE proteins and their role in the environmental control of leaf vein pattern

植物暴露在不断变化的环境中，无法逃脱。相反，它们改变了发展，生产出一种更适合新环境的形式。激素生长素控制着植物发育中的许多重要事件，如确定叶子在哪里形成，枝条和根枝在哪里，以及将根转向重力。生长素的定向转运取决于PINFORMED(PIN)转运蛋白在细胞膜特定一侧的极性定位。植物生长的许多环境反应是通过改变PIN的定位从而改变细胞的极性、组织的生长和组织来实现的。我对叶脉的形成和组织感兴趣，这也取决于PIN在细胞内的极性定位，这些细胞将成为叶脉。我的团队发现了FORKED1(FKD1)基因，它对在这些细胞中定位PIN非常重要。如果基因发生突变，PIN永远不会定位在细胞的顶端，静脉不会相遇形成正常的闭合环，而是形成一个开放的、“分叉”的静脉图案。我们发现FKD1在分泌途径中发挥作用，将PIN蛋白带到质膜上。我建议使用FKD1来研究将蛋白质定向到细胞顶端的其他因素，这是一种以前提出的、鲜为人知的途径。FKD1是植物特有的基因家族，存在于所有植物基因组中。该基因最先出现在非维管束苔类植物中，表明其功能早于血管。一个突变体敲除了拟南芥基因家族的四个成员，它的根很短，对重力没有反应，叶子畸形、结块。显微镜观察发现，隆起和凹陷中含有一片片随机取向的细胞。叶片中的细胞通常是极化的，其长端平行于中脉。虽然植物学家提出生长素可能会使叶片内的细胞两极分化，但还没有人能够在实验中测试这一想法。这些突变体为探索叶细胞极化的机制提供了独特的工具。静脉图案会随着环境的变化而变化。例如，干旱条件会导致更高的叶脉密度和更多的节理，这是一种有助于植物保持光合作用的适应。有趣的是，基因家族的成员都有受干旱和应激激素ABA调节的序列，我的学生们已经证明FKD1对ABA有反应。此外，FKD1或其他基因家族成员的突变也会消除对干旱的静脉模式反应。因此，我提出，该基因家族通过包括ABA在内的压力信号通路进行调节，从而允许静脉模式对压力进行适应，并设计了实验来验证这一观点。通过揭示实现PIN局部化和细胞极性的新过程，并定义环境如何影响这些过程，我的研究将允许操纵更适合环境的植物形态。

Plants are exposed to a constantly changing environment from which they cannot escape. Instead, they alter development to produce a form better adapted to the new environment. The hormone auxin controls many important events in plant development such as defining where leaves form, where shoots and root branch and turning roots towards gravity. Auxin is directionally transported from cell to cell dependent upon the polar localization of PINFORMED (PIN) transport proteins to a particular side of the cell membrane. Many of the environmental responses of plant growth are achieved by changing PIN localization and hence cell polarity, tissue growth and organization. I am interested in the formation and organization of leaf veins, which also depends on polar localization of PIN within cells that will become veins. My group discovered the FORKED1 (FKD1) gene, that is important for localizing PIN within these cells. If the gene is mutated, PIN is never localized to the top side of cells, and rather than meeting to form the normal closed loops, the veins form an open, “forking” vein pattern. We found that FKD1 works in the secretory pathway, taking the PIN protein to the plasma membrane. I propose to use FKD1 to study other factors that direct proteins to the top side of cells, a previously proposed pathway that is little understood. FKD1 is in a gene family unique to plants and found in all plant genomes. The gene first appears in non-vascular liverworts, indicating that its function predates veins. A mutant that knocks out four members of the Arabidopsis gene family has short roots that do not respond to gravity and misshapen, lumpy leaves. Microscopic observation reveals that the bumps and hollows contain patches of randomly oriented cells. Cells in leaves are normally polarized with their long ends parallel to the midvein. While botanists have suggested that auxin might polarize the cells within the leaf, no one has been able to test the idea experimentally. The mutants provide a unique tool to explore the mechanism by which leaf cells are polarized. Vein pattern changes in response to the environment. For example, drought conditions cause a higher vein density with more joints, an adaptation that helps plants maintain photosynthesis. Interestingly, members of the gene family have sequences that suggest regulation by drought and the stress hormone ABA, and my students have shown that FKD1 responds to ABA. As well, mutation to FKD1 or other gene family members eliminates the vein pattern response to drought. Therefore, I propose that the gene family allows vein pattern adaptation to stress, through its regulation by stress-signaling pathways including ABA, and have designed experiments to test this idea. By revealing novel processes that achieve PIN localization and cell polarity and defining how the environment influences these processes, my research will allow manipulation of plant form better suited to the environment.