Alfalfa (Medicago sativa L.) pho2 mutant plants hyperaccumulate phosphate

Authors: MILLER, SUSAN - Retired ARS Employee; Dornbusch, Melinda - Mindy; FARMER, ANDREW - National Center For Genome Research; HUERTAS, RAUL - The James Hutton Institute; GUTIERREZ-GONZALEZ, JUAN - University Of Leon; YOUNG, NEVIN - University Of Minnesota; Samac, Deborah - Debby; Curtin, Shaun

Submitted to: G3, Genes/Genomes/Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/12/2022
Publication Date: 4/26/2022
Citation: Miller, S.M., Dornbusch, M.R., Farmer, A., Huertas, R., Gutierrez-Gonzalez, J.J., Young, N.D., Samac, D.A., Curtin, S.J. 2022. Alfalfa (Medicago sativa L.) pho2 mutant plants hyperaccumulate phosphate. G3, Genes/Genomes/Genetics. 12(6). Article jkac096. https://doi.org/10.1093/g3journal/jkac096.
DOI: https://doi.org/10.1093/g3journal/jkac096

Interpretive Summary: Concentrated livestock farming systems and dense urban populations are major point sources of phosphate (P) run-off into rivers and lakes. Excessive P accelerates eutrophication of waterways, reducing water quality and ecological function, and contributes to the annual “dead zone” in the Gulf of Mexico. Disposal of animal manure on crop land reduces point sources of pollution and growing crops on these lands can reduce P, but many soils retain excessive levels of P. New methods are needed for protecting waterways, remediating high P soils, and reclaiming P for use on crops. A set of novel alfalfa plants with mutations in a phosphate uptake regulatory gene were generated by gene editing methods. Alfalfa has two forms of this gene for a total of eight copies, making gene editing of all copies challenging. Using novel methods to identify mutants, plants were identified with mutations in all copies and the mutations were inherited in the next generation. P accumulation in mutated plants increased with the number of mutated genes up to 10-fold higher than the wild-type plant. This alfalfa germplasm will have practical applications for reclaiming P from contaminated or enriched soil and plants can be deployed near waterways to protect ecosystems from eutrophication.

Technical Abstract: In this manuscript we describe a set of novel alfalfa (Medicago sativa) plants that hyper-accumulate Phosphate ion (Pi) at levels five to ten-fold higher than wild-type. This alfalfa germplasm will have practical applications reclaiming Pi from contaminated or enriched soil or in conservation buffer strips to protect waterways from Pi run-off. Hyper-accumulating alfalfa plants were generated by targeted mutagenesis of PHOSPHATE2 (PHO2) using newly created CRISPR/Cas9 reagents and an improved mutant screening strategy. PHO2 is a UBC24 that encodes a ubiquitin conjugating E2 enzyme previously characterized in Arabidopsis thaliana, M. truncatula and Oryza sativa. Mutations of PHO2 disrupt Pi homeostasis resulting in Pi hyper-accumulation. Successful CRISPR/Cas9 editing of PHO2 demonstrates that this is an efficient mutagenesis tool in alfalfa despite its complex autotetraploid genome structure. Arabidopsis and M. truncatula ortholog genes were used to identify haplo-alleles in outcrossing tetraploid M. sativa with the aim of generating heritable mutations in the two PHO2-like genes of alfalfa (MsPHO2-1 and MsPHO2-2). After delivery of the reagent and regeneration of shoots from transformed leaf explants, plants with mutations in all four haplo-alleles of MsPHO2-1 and MsPHO2-2 could be identified. These plants were evaluated for morphology, Pi accumulation, heritable transmission of targeted mutations, segregation of mutant haplo-alleles and removal of T-DNA(s). The Agrobacterium-mediated transformation assay and gene editing reagents reported here were also evaluated for further optimization in the context of improved alfalfa functional genomic studies.