Skip to main content

Robustness and specialization among hormone transporters: Redundant and unique roles

Periodic Reporting for period 2 - RobustHormoneTrans (Robustness and specialization among hormone transporters: Redundant and unique roles)

Reporting period: 2019-07-01 to 2020-12-31

Plant growth and development is governed by finely tuned, highly regulated hormone gradients. Impressive progress has been made in understanding plant hormone signaling, but knowledge on the mechanisms underlying their precise localization at the tissue and subcellular levels is still very limited. We and others have identified the first bona fide GA transporters in plants as members of the NPF protein family. Proteins from the ABC family were shown to transport the CK, ABA, and auxin hormones. Although these studies suggested specialized functions for members of these large protein families, progress in understanding their level of specialization has been limited by the scarcity of loss-of-function phenotypes, masked by the highly redundant plant genome.
The goal of this proposal is to reveal the robust and specialized function of the NPF and ABC plant hormone transporter families. The project places key technological challenges that require multi-disciplinary expertise to examine how plants balance redundancy and specialization to tightly regulate hormone localization. Broad and targeted transportome screens using multi-targeted artificial miRNAs and CRISPR technology in Arabidopsis and tomato, respectively, are designed to unveil novel plant hormone transporters, with an emphasis on subcellular localized transporters and the missing GA exporters. Specialization and robustness of candidate transporters are evaluated by integrating in vitro transport assays with in vivo growth and development experiments.
1) We have performed whole plant and cellular transportome amiRNA genetic screens to identify redundant hormone transporters in Arabidopsis.
2) We have engineered CRISPR-based multiplex populations to identify redundant hormone transporter families in Arabidopsis and tomato.
3) We have dissected the contributions of specialization and robustness to hormone transporter functions.
4) We are developing a genetic system that will allow to reveal the hormone subcellular map
The funded work is aimed to develop the frontiers of genetic screens. During the past two decades, genetic variation and forward genetics screen have been expanded by creating random mutagenized lines using chemical or radiation treatments leading to the identification of novel genetic processes. However, these approaches cannot overcome the genetic redundancy problem. Here we generate a unique targeted forward-genetic approach that bypasses functional redundancy in plants with a dynamic screening range. The amiRNA and CRISPR genomic populations that generated are designed in a flexible manner to enable targeted screening for gene families, ranging from hormone transporters to the entire genome. The unique multi-targeting populations generated here serve as groundbreaking reagents that will enable many plant genetics studies. Finally, the genetic toolbox constructed here could be used in breeding crop programs to improve agricultural traits.

In addition, we have created hormone subcellular maps. Since multiple lines of evidence demonstrate that bioactive hormones, their intermediates, and conjugates are regulated at the subcellular level, we constructed a genetically based method that allows the isolation of different plant organelles. We utilize our unique method to draw the first hormone subcellular maps in plants. Results from this project provide researches worldwide with detailed quantitative information regarding subcellular localization of active hormones, their intermediates, and conjugates. Beyond the scientific knowledge obtained in this project, our proposed method to isolate different plant organelles enables subcellular transcriptomics, metabolomics, and proteomics research.