Identification and functional characterization of the sugarcane (Saccharum spp.) AMT2-type ammonium transporter ScAMT3;3 revealed a presumed role in shoot ammonium remobilization

Rodolfo A Maniero; Alessandra Koltun; Marielle Vitti; Bruna G Factor; Nathalia de Setta; Amanda S Câmara; Joni E Lima; Antonio Figueira

doi:10.3389/fpls.2023.1299025

Identification and functional characterization of the sugarcane (Saccharum spp.) AMT2-type ammonium transporter ScAMT3;3 revealed a presumed role in shoot ammonium remobilization

Front Plant Sci. 2023 Nov 30:14:1299025. doi: 10.3389/fpls.2023.1299025. eCollection 2023.

Authors

Rodolfo A Maniero^#¹, Alessandra Koltun^#¹, Marielle Vitti¹, Bruna G Factor¹, Nathalia de Setta², Amanda S Câmara³, Joni E Lima⁴, Antonio Figueira¹

Affiliations

¹ Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Piracicaba, SP, Brazil.
² Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, São Bernardo do Campo, SP, Brazil.
³ Genebank Department, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Seeland, Germany.
⁴ Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

^# Contributed equally.

Abstract

Sugarcane (Saccharum spp.) is an important crop for sugar and bioethanol production worldwide. To maintain and increase sugarcane yields in marginal areas, the use of nitrogen (N) fertilizers is essential, but N overuse may result in the leaching of reactive N to the natural environment. Despite the importance of N in sugarcane production, little is known about the molecular mechanisms involved in N homeostasis in this crop, particularly regarding ammonium (NH₄ ⁺), the sugarcane's preferred source of N. Here, using a sugarcane bacterial artificial chromosome (BAC) library and a series of in silico analyses, we identified an AMMONIUM TRANSPORTER (AMT) from the AMT2 subfamily, sugarcane AMMONIUM TRANSPORTER 3;3 (ScAMT3;3), which is constitutively and highly expressed in young and mature leaves. To characterize its biochemical function, we ectopically expressed ScAMT3;3 in heterologous systems (Saccharomyces cerevisiae and Arabidopsis thaliana). The complementation of triple mep mutant yeast demonstrated that ScAMT3;3 is functional for NH₃/H⁺ cotransport at high availability of NH₄ ⁺ and under physiological pH conditions. The ectopic expression of ScAMT3;3 in the Arabidopsis quadruple AMT knockout mutant restored the transport capacity of ¹⁵N-NH₄ ⁺ in roots and plant growth under specific N availability conditions, confirming the role of ScAMT3;3 in NH₄ ⁺ transport in planta. Our results indicate that ScAMT3;3 belongs to the low-affinity transport system (K_m 270.9 µM; V_max 209.3 µmol g^-1 root DW h^-1). We were able to infer that ScAMT3;3 plays a presumed role in NH₄ ⁺ source-sink remobilization in the shoots via phloem loading. These findings help to shed light on the functionality of a novel AMT2-type protein and provide bases for future research focusing on the improvement of sugarcane yield and N use efficiency.

Keywords: Arabidopsis thaliana, heterologous expression; Saccharomyces cerevisiae; low-affinity transport system; nitrogen; nitrogen use efficiency.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was financed by FAPESP (The São Paulo Research Foundation) through Regular Research Grants (2016/14669-8; 2013/15989-8), as well as fellowships awarded to JL (2010/11313-1) and RM (2017/00460-2). Additional support was provided by CNPq (Brazilian National Council for Scientific and Technological Development) and CAPES (Coordination for the Improvement of Higher Education Personnel). AF is currently the recipient of a CNPq research fellowship (310645/2021-2). Publication fees were covered by the Universidade de São Paulo and FEALQ.