Developmental Control and Significance of knotted1 Intercellular Trafficking

多细胞生物体的发展需要对蛋白质的活性和定位进行仔细的调节。随着基因组计划开始揭示建造植物所需的基因组合，科学家们现在面临着基因在哪里、何时表达以及蛋白质在哪里定位的复杂问题。对这种复杂性施加的是这样一个事实，即特定的植物基因产物通过称为胞间连丝的特殊通道在细胞之间进行运输，因此它们的最终位置可能距离它们制造的细胞很远。具体地说，已知一些发育中的转录因子在细胞之间进行运输，尽管人们对这一过程的发育意义和调控知之甚少。本项目以KNOTTED1(KN1)基因为模型系统，旨在了解植物蛋白细胞间转运的发育规律。显性Kn1突变体对玉米叶片细胞命运具有非自主性影响，显微注射分析表明KN1蛋白和mRNA可以通过胞间连丝在叶片细胞之间运输。这些分析受到可作为靶向的组织类型的限制，限制了提出与KN1贩运在其干细胞维持功能中的作用有关的发育相关问题的能力。因此，KN1与绿色荧光蛋白的蛋白质融合被开发出来，并用于观察在完整植物组织中的体内运输。在未来的研究中，这些研究将被扩展到提出关于分生组织中胞间连丝运输的控制的重要问题。具体地说，将估计分生组织中胞间连丝的大小排除限制，并将测试是否存在用于蛋白质运输的共质结构域。KN1中的贩运信号序列也将被本地化。这项研究将有助于理解植物中细胞间的通讯。它还将增加胞间连丝的基本知识，胞间连丝在分子水平上的特征很差。胞间连丝对于光合作用产物的分配、植物的形态形成和抵御病原菌具有重要作用。因此，这些关于蛋白质运输的研究可能对提高农业生产力具有重大意义。

The development of a multicellular organism requires careful regulation of protein activity and localization. As genome projects are beginning to reveal the complement of genes required to build a plant, scientists now face the complex issue of where and when genes are expressed and where proteins are localized. Imposed on this complexity is the fact that specific plant gene products traffic between cells through specialized channels called plasmodesmata, so their final location may be distant from the cell where they are made. Specifically, some developmental transcription factors are known to traffic between cells, though little is known of the developmental significance and regulation of this process. This project aims to understand the developmental regulation of cell to cell trafficking of plant proteins, using the KNOTTED1 (KN1) gene as a model system. Dominant Kn1 mutants have non-autonomous effects on cell fate in the maize leaf, and using microinjection assays it was shown that KN1 protein and mRNA can traffic between leaf cells, through plasmodesmata. These assays were constrained by the type of tissue that could be targeted, limiting the ability to ask developmentally relevant questions pertaining to the role of KN1 trafficking in its function for stem cell maintenance. Protein fusions of KN1 to the green fluorescent protein were therefore developed and used to visualize trafficking in vivo in intact plant tissues. In the future research, these studies will be extended to ask significant questions about control of plasmodesmal trafficking in the meristem. Specifically, the size exclusion limit of plasmodesmata in the meristem will be estimated, and the experiments will test if symplasmic domains for protein transport exist. Trafficking signal sequences in KN1 will also be localized. The study will contribute to the understanding of cell to cell communication in plants. It will also increase basic knowledge of plasmodesmata, which are poorly characterized at the molecular level. Plasmodesmata are important for the allocation of photosynthetic products, for plant morphogenesis and for defense against pathogens. Therefore these studies of protein trafficking could have significant implications for improving agricultural productivity.