Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

CRISPR Activation of Poplar Target of Rapamycin Genes Improves Nitrogen Use Efficiency and Indicates Possible Functional Divergence

Authors:

Reuben Tayengwa1*, Anil Kumar1, Gen Li1, Yiping Qi1, Edward Eisenstein1, Victor Busov2, Hairong Wei2, Gary Coleman1

Institutions:

1University of Maryland; 2Michigan Technological University

Goals

The goal of this research is to determine the role of signaling mediated by the target of rapamycin complex 1 (TORC1) in nutrient sensing in poplar and elucidate the functional role of genes regulating nutritional responses using CRISPR gene editing, genomics, biochemical, and computational approaches.

Abstract

Poplar (Populus spp.) is an important and sustainable bioenergy and bioproduct plant feedstock, yet scientists’ understanding of the pathways and networks governing resource use efficiency is poorly developed. The protein target of rapamycin (TOR) kinase is part of an evolutionally conserved central hub that integrates nutrient, energy, hormones, biotic, and abiotic stresses signals by regulating transcription, translation, and metabolism. Compared to other plants species, such as Arabidopsis and rice, poplar contains two TOR genes. Using genome editing and computation approaches, this project will elucidate the role of the two poplar TOR genes in nitrogen utilization and if the two TOR genes have diverged in function.

An analysis of a compendium (over 800) of publicly available poplar RNA sequencing (RNA-seq) datasets indicates that both TOR genes are expressed in a variety of tissues and conditions. TOR expression appears to be coordinately regulated with the interacting partners LST8 and regulatory-associated protein of TOR (RAPTOR). Studies with each of the two TOR promoter (2.5 kilobyte) sequences fused to a beta-glucuronidase, green fluorescent protein (GUS-GFP) reporter gene indicate overlapping patterns of expression for each TOR gene. Using CRISPR activation of each TOR gene individually or the simultaneous activation of both poplar TOR genes showed that TOR activation enhanced growth under suboptimal levels of nitrogen fertility. This enhanced growth included significantly greater height, leaf area, stem dry weight, leaf dry weight and above ground biomass. Growth analysis suggests that the increase in growth was related to internode initiation/production as opposed to internode elongation. CRISPR TOR activation also revealed phenotypic differences resulting from the activation of the two poplar TOR genes. Interestingly, simultaneous activation of both TOR genes did not result in increased growth or biomass production. The team hypothesizes that differences in the ratio of homo and hetero dimers of TOR may result in different outputs that affects growth, biomass production, and phenotype. This hypothesis is being tested using genome edited poplars with independent biallelic mutants for each TOR gene; using CRISPR-Combo gene edited poplars for the simultaneous production of biallelic knockout mutants of one TOR gene while the respective second TOR gene is activated; and production of an expression variants via independent promoter editing of the two poplar TOR genes. Additionally, both constitutive active and dominant negative versions of poplar rho of plants (ROP) genes corresponding to ROP2 and ROP4 have been generated and transformed into poplar and the effect on nutrient use efficiency and root growth will be presented.

Funding Information

This research was supported by the DOE Office of Science, BER, grant no. DE-SC0023011.