An miR156-regulated nucleobase-ascorbate transporter 2 confers cadmium tolerance via enhanced anti-oxidative capacity in barley

Introduction: Cadmium (Cd) is one of the most detrimental heavy metal pollutants, seriously affecting crop production and human health. Nucleobase-ascorbic acid transporters (NAT) are widely present in many living organisms including plants, animals and microbes; however, the role of NAT in plant Cd tol-erance remains unknown. Objectives: To identify Cd-induced miRNAs that target HvNAT2 and to determine the role of this gene and its product in Cd tolerance. Methods: High-throughput-sequencing was used to identify the miRNA expression profile of barley roots in response to Cd stress. Overexpression (OX) and RNAi lines were then constructed for HvNAT2 and com-parative transcriptomic analysis was performed to determine the function of this transporter examining its effects on traits such as Cd uptake/flux and translocation, morphology and antioxidant capacity in relation to Cd tolerance. In addition, phylogenetic analysis was performed to obtain insights into the evolution of HvNAT2. Results: Cd stress-induced genome-wide expression profiles of miRNAs identified a Cd-induced miRNA, miR156g-3p_3, that had HvNAT2 as its target. HvNAT2 was negatively regulated in the high-Cd-accumulating and Cd-tolerant genotype Zhenong8. Evolutionary analysis indicated that orthologues of the plasma membrane localized, HvNAT2, can be traced back to the sister group of land plants, the strep-tophyte algae. Overexpression of HvNAT2 increases Cd tolerance with higher tissue Cd accumulation but less oxidative damage in transgenic barley plants. RNAi of HvNAT2 leads to a significant reduction of Cd tolerance. The higher Cd accumulation in roots of the OX3 line was also demonstrated by confocal micro-scopy and electrophysiology. Transcriptome analysis showed that the enhancement of antioxidant capac-ity by HvNAT2 was related to stress signaling pathways. Furthermore, oxidative stress tolerance in HvNAT2-OX plants was regulated by the synthesis of phytochelatins and the glutathione metabolism cycle. Conclusion: Our study reveals a key molecular mechanism of NAT in Cd tolerance in plants that is useful for sustainable agricultural production and management of hazardous this heavy metal for better envi-ronment management and ecosystem function. (c) 2022 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Automated summary

Cadmium (Cd) is a detrimental heavy metal pollutant that has negative effects on crop production and human health. This study identified a Cd-induced miRNA, miR156g-3p_3, that targets HvNAT2 and found that overexpression of HvNAT2 enhances Cd tolerance and reduces oxidative damage in transgenic barley plants. This research provides valuable insights into the molecular mechanism of NAT in Cd tolerance and has implications for sustainable agricultural production and environmental management.