Functions and signaling mechanisms of a non-proteinogenic amino acid ACC: the case for a novel plant hormone

植物产生的激素，如乙烯，基本上共同控制着植物生物学的所有方面。这个项目提出了一个新的假设，即植物用来制造乙烯的小分子本身就是一种影响植物生长和发育的新型植物激素，并进一步研究了这种激素在整个植物进化过程中如何在分子水平上发挥作用。该项目有可能改变现有的教条，需要重新评估数十年的乙烯文献，并影响未来乙烯实验的进行方式，从而有可能改变植物激素领域。从这一项目中获得的知识可有助于今后为造福社会而修改农作物的战略。该项目通过每年在首席调查员的实验室提供两到三个学生实习机会，扩大了代表性不足的学生的参与。实习涉及与霍华德大学(一所历史上的黑人学院或大学)和埃莉诺·罗斯福高中(一所当地的公立学校，超过70%的学生是少数族裔代表不足)的合作。实习生执行项目的各个方面，同时接受科学方法、数据分析和沟通方面的宝贵培训。该项目还为两名博士后科学家、两名研究生和两名马里兰大学本科生提供培训和职业准备。为了进一步扩大参与，首席研究员与霍华德大学本科生共同教授暑期研究工作坊。众所周知，乙烯生物合成途径中的乙烯前体是1-氨基环丙烷-1-羧酸(ACC)，这是一种非蛋白生成氨基酸。令人信服的新数据表明，ACC本身可能是一个重要的信号分子，在进化上先于高等陆地植物有效地将ACC转化为乙烯的能力。特别是，ACC抑制多形地钱的细胞分化和生长。本项目通过对ACC功能的分析，包括对ACC信号转导机制的研究，验证了ACC是一种新的植物激素的假设。对Acc功能的深入研究是通过对Marchantia以及植物谱系中进化相关的物种(Chlamydomonas reinhardtii，Spirogyra pratensis，Physcomitrella patens和Arabiopsis thaliana)的ACC合成突变体和ACC反应的研究来提供的，从而解决了ACC功能的保守，同时利用了基础植物中较低的基因拷贝数。在拟南芥中，初步的研究结果表明，ACC可以诱导花粉管的生长，同时伴随着钙离子的内流，并能刺激初生根的生长。ACC激活拟南芥根原生质体内的钙电流，而且这种激活依赖于谷氨酸受体样离子通道。该项目结合了分子遗传学、膜片钳技术、离子特异性振动探针和钙离子实时成像技术，对ACC是GLR配体的假设进行了验证。通过对GLR突变体和ACC合成突变体在植物物种范围内的表型比较，寻找遗传证据。ACC激活GLRs，进而调控花粉管生长和/或根生长中的钙信号，将是理解ACC信号和GLR信号机制的一个突破性进展，代表了目前植物中尚无共识的钙离子调节的配基调控系统。在Marchantia中发现的惊人的ACC反应为ACC信号突变体提供了遗传筛选，随后是基于T-DNA标签或全基因组测序的基因克隆。

Plants produce hormones such as ethylene that collectively control essentially all aspects of plant biology. This project advances a new hypothesis that a small molecule, which plants use to make ethylene, functions itself as a novel plant hormone that impacts plant growth and development, and further investigates how the hormone functions at the molecular level throughout the evolutionary breadth of plants. The project has the potential to transform the plant hormone field by altering existing dogma, by requiring the reevaluation of several decades of ethylene literature and by impacting how future ethylene experiments are carried out. The knowledge gained from this project could contribute to future strategies for modifying crop plants for the benefit of society. The project broadens participation of underrepresented students by providing two to three student internships per year in the lab of the principal investigator. The internships involve partnerships with Howard University (an Historically Black College or University) and Eleanor Roosevelt High School (a local public school in which over 70% of the students are underrepresented minorities). The interns carry out aspects of the project while receiving valuable training in scientific methods, data analysis and communication. The project also provides training and career preparation for two postdoctoral scientists, two graduate students and two University of Maryland undergraduates. To further broaden participation, the principal investigator co-teaches summer research workshops for Howard University undergraduates. The well-known ethylene precursor in the ethylene biosynthesis pathway is 1-aminocyclopropane-1-carboxylic acid (ACC), a non-proteinogenic amino acid. Compelling new data suggest that ACC itself may be an important signaling molecule that evolutionarily predated the ability of higher land plants to efficiently convert ACC to ethylene. In particular, ACC inhibits cellular differentiation and growth in the liverwort Marchantia polymorpha. This project tests the hypothesis that ACC is a novel plant hormone through analyses of ACC function, including an investigation of ACC signaling mechanisms. Insight into ACC function is provided by studies of ACC synthesis mutants and ACC responses in Marchantia, and in evolutionarily relevant species in the plant lineage (Chlamydomonas reinhardtii, Spirogyra pratensis, Physcomitrella patens and Arabidopsis thaliana), thus addressing the conservation of ACC function, while taking advantage of lower gene copy number in basal plants. In Arabidopsis, preliminary findings indicate that ACC can induce pollen tube growth concomitant with Ca2+ influx and can stimulate primary root growth. ACC activates Ca2+ currents in Arabidopsis root protoplasts, and notably, this activation is dependent on glutamate receptor-like (GLR) ionotropic channels. The project tests the hypothesis that ACC is a GLR ligand using a combination of molecular genetics, patch-clamping, ion-specific vibrating probes and live imaging of Ca2+. Genetic evidence is sought based on phenotypic comparisons of glr mutants and ACC synthesis mutants in the range of plant species. The activation of GLRs by ACC, leading to the regulation of Ca2+ signaling in pollen tube growth and/or root growth, would be a breakthrough in understanding mechanisms of ACC signaling, as well as GLR signaling, and would represent a ligand-operated system of Ca2+ regulation for which no consensual system currently exists in plants. The striking ACC response uncovered in Marchantia provides for a genetic screen for ACC signaling mutants, followed by gene cloning based on T-DNA tagging or whole genome sequencing.