Molecular Characterization of AtCam9

项目摘要：钙离子（Ca2+）作为信使在真核细胞中触发对内外刺激（如激素和环境应激）的生理反应。Ca2+的初始感知是由特异性结合金属的蛋白质完成的，并反过来调节其他细胞蛋白质的活动。这类蛋白最典型的例子是钙调蛋白（Cam）。Cam蛋白长期以来一直参与调节植物的生长发育以及植物对应激刺激的反应，但它们参与这些反应的具体机制尚不清楚。本研究的重点是确定拟南芥中Ca2+结合蛋白AtCam基因家族的新成员ATCam9的作用。初步数据显示，AtCam9的功能机制可能与更传统的Cam蛋白相似，但不完全相同。这表明AtCam9通过与一组独特的蛋白质相互作用并调节其功能。此外，与对照拟南芥植株相比，AtCam9基因敲除突变体表现出快速生长/整体放大的表型；敲除常规Cam基因会产生生长表型降低的植株。因此，AtCam9似乎在分子和有机体水平上以一种与传统Cam蛋白不同的方式起作用。在这个提议中将要测试的假设是AtCam9是否作为植物生长速率的负调节因子。为了测试该模型并确定AtCam9调节生长的途径，该项目将追求四个具体目标：(1)将确定AtCam9是否通过调节细胞扩增来调节生长，或者它是否也在细胞分裂中发挥作用。(2)确定常规的AtCam蛋白是否可以替代AtCam9，反之，确定AtCam9是否可以替代常规的Cam蛋白。(3)确定AtCam9表达的细胞类型和亚细胞位置。作为这一目标的一部分，将确定AtCam9是否自由扩散，这一特性将表明AtCam9是否通过与其他蛋白质相互作用而起作用。(4)将确定AtCam9相互作用蛋白的身份，并确定与AtCam9相互作用的时间和亚细胞位置。建议活动的更广泛影响。阐明由AtCam9介导的特定功能将有助于更深入地了解Ca2+在调节植物生长和发育中的作用，这些因素对农业生产力至关重要。了解AtCam9基因敲除植物表现出的意外表型的分子基础，可能有助于设计或控制植物生长的新策略。该项目将为本科生到博士后提供蛋白质生物化学、拟南芥分子遗传学、植物生长分析和细胞成像等方面的培训。

Project Abstract Calcium ions (Ca2+) are used as messengers in all eukaryotic cells to trigger physiological responses to internal and external stimuli, such as hormones and environmental stresses. The initial perception of Ca2+ is accomplished by proteins that specifically bind the metal and in turn modulate the activities of other cellular proteins. The best characterized example of this class of proteins is calmodulin (Cam). Cam proteins have long been implicated in regulating plant growth and development as well as responses of plants to stressful stimuli, but the specific mechanisms by which they participate in these responses are not well understood. This proposal focuses on defining the role of ATCam9, a novel member of the AtCam gene family of Ca2+-binding proteins in Arabidopsis. Preliminary data have revealed that AtCam9 might function by a mechanism similar to, but not identical with, more conventional Cam proteins. This suggests that AtCam9 functions by interacting with and regulating a unique set of proteins. In addition, knockout mutants of AtCam9 display a rapid growth/overall enlarged phenotype compared with control Arabidopsis plants; knockouts of conventional Cam genes yield plants with reduced growth phenotypes. Thus, AtCam9 appears to function in a manner distinct from conventional Cam proteins on both the molecular and organismic levels. The hypothesis that will be tested in this proposal is whether AtCam9 functions as a negative regulator of the rate of plant growth. To test this model and define the pathway(s) by which AtCam9 modulates growth, the project will pursue four specific aims:(1) It will be determined whether AtCam9 regulates growth by modulating cell expansion or whether it also plays a role in cell division. (2) It will be determined whether conventional AtCam proteins can substitute for AtCam9, and conversely, whether AtCam9 can substitute for conventional Cam proteins. (3) The cell types and subcellular locations in which AtCam9 is expressed will be defined. As part of this aim, it will be determined whether AtCam9 is freely diffusible, a property that will indicate whether AtCam9 functions by interacting with other proteins. (4) The identities of AtCam9-interacting proteins will be determined, and the timing and subcellular locations of the interactions with AtCam9 will be defined.Broader Impact of the Proposed Activity. Elucidating the specific functions mediated by AtCam9 will provide a deeper understanding of the roles of Ca2+ in regulating plant growth and development, factors that are central to agricultural productivity. Understanding the molecular bases for the unexpected phenotype displayed by AtCam9 knockout plants may facilitate novel strategies for engineering or controlling plant growth. The project will provide training for students from the undergraduate through the postdoctoral levels in protein biochemistry, Arabidopsis molecular genetics, analyses of plant growth and cellular imaging.