A Toolkit for High Resolution Imaging of Cell Division and Phytohormone Signaling in Legume Roots and Root Nodules.

Marcin Nadzieja,Jens Stougaard,Dugald Reid

doi:10.3389/fpls.2019.01000

Abstract

Legume plants benefit from a nitrogen-fixing symbiosis in association with rhizobia hosted in specialized root nodules. Formation of root nodules is initiated by de novo organogenesis and coordinated infection of these developing lateral root organs by rhizobia. Both bacterial infection and nodule organogenesis involve cell cycle activation and regulation by auxin and cytokinin is tightly integrated in the process. To characterize the hormone dynamics and cell division patterns with cellular resolution during nodulation, sensitive and specific sensors suited for imaging of multicellular tissues are required. Here we report a modular toolkit, optimized in the model legume Lotus japonicus, for use in legume roots and root nodules. This toolkit includes synthetic transcriptional reporters for auxin and cytokinin, auxin accumulation sensors and cell cycle progression markers optimized for fluorescent and bright field microscopy. The developed vectors allow for efficient one-step assembly of multiple units using the GoldenGate cloning system. Applied together with a fluorescence-compatible clearing approach, these reporters improve imaging depth and facilitate fluorescence examination in legume roots. We additionally evaluate the utility of the dynamic gravitropic root response in altering the timing and location of auxin accumulation and nodule emergence. We show that alteration of auxin distribution in roots allows for preferential nodule emergence at the outer side of the bend corresponding to a region of high auxin signaling capacity. The presented tools and procedures open new possibilities for comparative mutant studies and for developing a more comprehensive understanding of legume-rhizobia interactions.

Highlights

Plant hormones control all aspects of plant development
We previously described TCSn activity upon rhizobial inoculation over initial steps of symbiosis (Reid et al, 2017), showing a broad early activation of cytokinin signaling in the root cortex of the plant
We previously described a sensitive auxin accumulation reporter, LjDII, for use in L. japonicus roots (Nadzieja et al, 2018)

Summary

INTRODUCTION

In legume plants, which enter into symbiotic relationship with soil bacteria called rhizobia, de novo formation of lateral organs called nodules must be coordinated with rhizobial infection for successful development of nitrogen fixing root nodules. These two separate, yet interdependent processes are regulated by plant hormones. Studying plant hormone dynamics in this context requires high resolution sensors in order to discriminate signaling that regulate nodule organogenesis and infection processes. We show that the fluorescent compatible clearing methodology provides a great improvement in imaging depth in multicellular tissues and enables comprehensive imaging of anatomical features and cell division during root and nodule development

RESULTS

DISCUSSION

MATERIALS AND METHODS