Functional characterization of the GhNRT2.1e gene reveals its significant role in improving nitrogen use efficiency in Gossypium hirsutum

Xinmiao Zhang; Jiajia Feng; Ruolin Zhao; Hailiang Cheng; Javaria Ashraf; Qiaolian Wang; Limin Lv; Youping Zhang; Guoli Song; Dongyun Zuo

doi:10.7717/peerj.15152

Functional characterization of the GhNRT2.1e gene reveals its significant role in improving nitrogen use efficiency in Gossypium hirsutum

PeerJ. 2023 Mar 28:11:e15152. doi: 10.7717/peerj.15152. eCollection 2023.

Authors

Affiliations

¹ State Key Laboratory of Cotton Biology, Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, Henan, China.
² Department of Plant Breeding and Genetics, Faculty of Agriculture and Environment, The Islamia University of Bahawalpur, Bahawalpur, Punjab, Pakistan.

Abstract

Background: Nitrate is the primary type of nitrogen available to plants, which is absorbed and transported by nitrate transporter 2 (NRT2) at low nitrate conditions.

Methods: Genome-wide identification of NRT2 genes in G. hirsutum was performed. Gene expression patterns were revealed using RNA-seq and qRT-PCR. Gene functions were characterized using overexpression in A. thaliana and silencing in G. hirsutum. Protein interactions were verified by yeast two-hybrid and luciferase complementation imaging (LCI) assays.

Results: We identified 14, 14, seven, and seven NRT2 proteins in G. hirsutum, G. barbadense, G. raimondii, and G. arboreum. Most NRT2 proteins were predicted in the plasma membrane. The NRT2 genes were classified into four distinct groups through evolutionary relationships, with members of the same group similar in conserved motifs and gene structure. The promoter regions of NRT2 genes included many elements related to growth regulation, phytohormones, and abiotic stresses. Tissue expression pattern results revealed that most GhNRT2 genes were specifically expressed in roots. Under low nitrate conditions, GhNRT2 genes exhibited different expression levels, with GhNRT2.1e being the most up-regulated. Arabidopsis plants overexpressing GhNRT2.1e exhibited increased biomass, nitrogen and nitrate accumulation, nitrogen uptake and utilization efficiency, nitrogen-metabolizing enzyme activity, and amino acid content under low nitrate conditions. In addition, GhNRT2.1e-silenced plants exhibited suppressed nitrate uptake and accumulation, hampered plant growth, affected nitrogen metabolism processes, and reduced tolerance to low nitrate. The results showed that GhNRT2.1e could promote nitrate uptake and transport under low nitrate conditions, thus effectively increasing nitrogen use efficiency (NUE). We found that GhNRT2.1e interacts with GhNAR2.1 by yeast two-hybrid and LCI assays.

Discussion: Our research lays the foundation to increase NUE and cultivate new cotton varieties with efficient nitrogen use.

Keywords: Expression pattern; Gene family; Gossypium hirsutum; High-affinity nitrate transporter; NRT2; NUE.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Arabidopsis* / genetics
Gossypium* / genetics
Nitrate Transporters
Nitrates / metabolism
Nitrogen / metabolism
Plant Proteins / genetics
Saccharomyces cerevisiae / metabolism

Substances

Plant Proteins
Nitrates
Nitrogen
Nitrate Transporters

Grants and funding

This work was supported by the National Natural Science Foundation of China (Grant No. 31621005 and Grant No. 31901581), the Central Public-interest Scientific Institution Basal Research Fund (Grant No. 1610162022054 and Grant No. 1610162022016) and the “Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.