Genome-Wide Characterization of the Wheat Pum Gene Family and Heterologous Functional Analysis of TaPum-III-5b in Arabidopsis Under Salt Stress.

Pumilio (Pum)-family proteins are evolutionarily conserved RNA-binding proteins that regulate post-transcriptional processes; however, their specific functions in staple crops, for example, wheat (Triticum aestivum), remain poorly understood. We identified 56 TaPum genes and classified them into three phylogenetic subgroups (I-III). The larger TaPum family size in allohexaploid wheat was associated with retention of A-, B-, and D-subgenome homoeologs, segmental duplication, and tandem duplication. After separating homoeologous pairs, 17 segmental duplication pairs and five tandem duplication pairs were identified, and ratios of nonsynonymous to synonymous substitutions (Ka/Ks) below 1 were consistent with purifying selection. Subgroup-specific motifs and the uneven distribution of cis-acting elements, particularly abscisic acid (ABA)- and methyl jasmonate (MeJA)-responsive elements, suggested potential divergence in promoter regulatory architecture. Transcriptomic profiling and independent quantitative reverse transcription-polymerase chain reaction analysis showed more consistent response of TaPum genes to salt stress than to cold or drought stress. Salt stress strongly promoted TaPum-III-5b expression, and its encoded protein exhibited nuclear and cytoplasmic localization. Heterologous overexpression of TaPum-III-5b in Arabidopsis thaliana increased salt tolerance, as indicated by enhanced survival; augmented peroxidase, superoxide dismutase, and catalase activities; and diminished accumulation of reactive oxygen species and malondialdehyde. Electrophoretic mobility shift assay (EMSA) demonstrated sequence-specific binding of TaDREB2B to a dehydration-responsive element (DRE)-containing sequence in the TaPum-III-5b promoter, whereas dual-luciferase (Dual-LUC) analysis showed activation of the wild-type promoter. Together, these results support a proposed regulatory framework in which TaPum-III-5b may contribute to salt-stress responses through as-yet-unidentified downstream RNA targets. This study characterizes the Pum gene family in wheat and identifies the TaDREB2B-TaPum-III-5b module as a candidate regulatory pathway associated with salt-stress responses. Its potential application in wheat improvement requires further validation in wheat.