Integrated miRNA-seq and RNA-seq analysis reveals stage-specific miRNA-mRNA regulatory networks in Populus...

Soil salinization is a major abiotic stress that severely constrains global forestry productivity. Plants deploy sophisticated gene regulatory networks for adaptation, in which microRNAs (miRNAs) have emerged as crucial post-transcriptional modulators. However, a comprehensive understanding of the dynamic and phase-specific roles of miRNAs in the salt stress response of perennial woody plants remains limited.

We performed an integrated miRNA-seq and RNA-seq analysis on Populus yunnanensis leaves across four treatment points: control (CK), short-term salt stress (T1) and long-term salt stress (T4), and recovery after stress (TR). Small RNA sequencing identified 571 miRNAs, including 339 known and 232 novel candidates, with expression dynamics highly sensitive to stress phases. Differential expression analysis revealed stage-specific miRNA repertoires across five biologically defined phases: Early Response (ER, T1vsCK; 6 DEMs), Long-term Adaptation (LA, T4vsCK; 17 DEMs), Recovered State (RS, TRvsCK; 17 DEMs), Stress Progression (SP, T4vsT1; 15 DEMs), and Recovery Process (RP, union of TRvsT1 and TRvsT4; 18 DEMs). Notably, ptc-miR6462 and ptc-miR6476 were unique to ER; ptc-miR169 was specific to SP; ptc-miR6457 was specific to RP; ptc-miR477 was unique to RS; while ptc-miR395 family members were active across all stages except ER. Concurrent transcriptomics unveiled extensive, duration-dependent transcriptional reprogramming, with DEGs increasing from 603 in ER to 3,027 in LA, and 2,239 DEGs persisting in RS. KEGG enrichment highlighted the central role of metabolic pathways across all stages, with phase-specific activation of signaling pathways (MAPK, plant hormone transduction) in LA and RS, membrane remodeling pathways (alpha-Linolenic acid metabolism) in RP, and sustained metabolic adjustments (cysteine and methionine metabolism, secondary metabolite biosynthesis) in RS. Integrated miRNA-mRNA network analysis constructed core regulatory circuits underpinning stage-specific adaptation, including ptc-miR395-APS1 (ATP sulfurylase 1) for antioxidant synthesis, ptc-miR319-MYB for growth-defense balance during recovery, and novel circuits involving ptc-miR6457b-MazG and ptc-miR6476-GINS, suggesting roles in nucleotide homeostasis and DNA replication protection.

Our study delineates a complex, phase-specific post-transcriptional regulatory network that orchestrates the salt stress adaptation of P. yunnanensis. By distinguishing five distinct phases-early response, long-term adaptation, stress progression, recovery process, and recovered state-we demonstrate that miRNAs function as precise temporal tuners, sequentially regulating distinct biological processes: from initial signal perception and DNA protection (ER), through sustained metabolic adaptation and antioxidant defense (LA, SP), to active recovery mechanisms (RP) and the establishment of stress memory (RS). These findings provide novel insights into the molecular basis of salt tolerance in trees and furnish valuable genetic resources for breeding stress-resilient forest varieties.