Small RNA Sequencing Revealed that miR4415, a Legume-Specific miRNA, was Involved in the Cold Acclimation of Ammopiptanthus nanus by Targeting an L-Ascorbate Oxidase Gene and Regulating the Redox State of Apoplast

Abstract

MicroRNAs (miRNAs) are small endogenous single-stranded RNAs that regulate plant growth, development, and environmental stress response posttranscriptionally. Ammopiptanthus nanus, a rare evergreen broad-leaved shrub in the temperate area of Central Asia, can tolerate freezing stress as low as –30 degrees centigrade in winter, and miRNA might be involved in the cold acclimation which enables A. nanus to obtain tolerance to freezing stress. Systematic identification and functional analysis of the miRNAs involved in the cold acclimation in A. nanus may promote understanding of the miRNA-mediated gene regulation network underlying cold acclimation. Here, based on small RNA and degradome sequencing, 256 miRNAs and 1,808 miRNA-target pairs were identified in A. nanus. A total of 39 cold-responsive miRNAs were identified, of which 29 were upregulated and ten were downregulated. These cold-responsive miRNAs may participate in the cold acclimation by regulating redox homeostasis (miR398, miR4415, and miR408), calcium signaling (miR5225 and miR5211), growth and development (miR159 and miR390), and small RNA–mediated gene silencing (miR168 and miR1515). We found that miR4415, a legume-specific miRNA, is involved in the cold acclimation of A. nanus by targeting an L-ascorbate oxidase gene and then regulating the redox state of the apoplast. Our study provides important data for understanding the regulatory role of miRNA in the cold acclimation of A. nanus.

Gene Profiling of the Ascorbate Oxidase Family Genes under Osmotic and Cold Stress Reveals the Role of AnAO5 in Cold Adaptation in Ammopiptanthus nanus

“…In previous studies, we proved that miR4415 was a legume-specific miRNA, which may have evolved independently after the emergence of a common ancestor of leguminous plants [ 38 ]. Interestingly, an AO-targeting miRNA, miR528, was also identified in rice, which was proven to be a monocotyledonous plant-specific miRNA [ 33 ], and the rice AO gene targeted by miR528 is an ortholog of AnAO5 ( Figure 8 A).…”

“…Multiple sequence alignment analysis showed that AnAO5 contained the three Pfam gene models for ascorbate oxidase and a transmembrane domain at the N-terminal. The subcellular localization study showed that AnAO5 was located in the apoplast [ 38 ]. The ortholog of AnAO5 in Arabidopsis is At4g39830, which was reported to encoded an apoplast localized AO.…”

“…As an apoplast-located AO, AnAO5 may participate in the response of plants to environmental stress and play other possible biological functions by regulating the redox balance of the apoplast via catalyzing the oxidation of AsA. In cold-acclimated A. nanus , the upregulation expression of AnAO5 gene led to decreased ASA content and increased DHA content, finally lowering the AsA/DHA ratio in apoplast, which was conducive to the accumulation of ROS [ 38 ]. It was reported that ROS, including H 2 O 2 and hydroxyl radicals, was involved in plant growth, development, and the response to environmental stress and, as a signal molecule, H 2 O 2 affected a variety of biological processes, including inducing stomatal closure, further enhancing the tolerance of plants to adverse external environments [ 14 , 17 ].…”

Multi-omics analysis reveals a link between Brassica -specific miR1885 and rapeseed tolerance to low temperature

“…At present, many MIRNA genes functioning in stress response have been identified by small RNA profiling and RNA-seq (Ahmed et al, 2020;Ma et al, 2022;Zhu et al, 2022). However, the MIRNA genes that function in the response to cold stress in crops remain largely unexplored.…”

Combined lncRNA and mRNA Expression Profiles Identified the lncRNA–miRNA–mRNA Modules Regulating the Cold Stress Response in Ammopiptanthus nanus

“…The chromosome-level reference genome of A. nanus has been reported and researchers have carried out a number of studies, using physiological and biochemical methods, transcriptomics, proteomics, and other omics techniques to analyze the abiotic stress tolerance mechanism and identify stress tolerance-related genes in A. nanus [ 26 , 27 , 28 , 29 , 30 ]. A recent study revealed that conserved and lineage-specific miRNAs contributed to the cold stress response by regulating ROS homeostasis and stress signaling by negatively regulating the corresponding targets [ 31 ]. Long non-coding RNAs may play important roles in the cold stress response in A. nanus by regulating mRNA/protein-coding genes.…”