Characterization of BrSTM16 from Brassica rapa and functional analysis of its role in regulating cold tolerance

The SHOOTMERISTEMLESS (STM) gene family controls shoot apical meristem homeostasis and participates in plant development and abiotic stress adaptation in higher plants. While the conserved roles of STM in maintaining meristem stability are well documented, the functions and regulatory mechanisms of BrSTM genes in Brassica rapa cold tolerance remain elusive, hindering our understanding of stress mediated meristem plasticity. Here, we systematically characterized the Brassica rapa STM gene family and identified 31 BrSTM members, all harboring the conserved KNOXⅠ domain and belonging to the class I KNOX family. Further functional analysis focused on the key candidate BrSTM16. Its promoter contains cold responsive and meristem specific regulatory elements, and the gene displays predominant expression in shoot apical meristems, with distinct tissue-specific and temperature dependent cold responses. Higher cold induction in cold-sensitive varieties indicates that BrSTM16 acts as a vital regulator linking meristem morphology and development to cold acclimation. Gene edited BrSTM16 mutants exhibited reduced survival rates and antioxidant enzyme activities under cold stress, confirming its positive role in regulating Brassica rapa cold tolerance. Transcriptome analysis suggested that BrSTM16 may modulate cold-induced metabolic homeostasis via the phenylpropanoid biosynthesis pathway. The disrupted expression of key pathway genes (PAL, 4CL, F5H) in knockout mutants was closely correlated with impaired cold resistance. Subcellular localization verified that BrSTM16 localizes to the nucleus. Six BrSTM16 interacting proteins with cold-responsive expression patterns were identified by yeast two-hybrid screening, and they participate in the BrSTM16 mediated cold-regulatory network. This study clarifies the characteristics of the BrSTM family and preliminarily reveals that BrSTM16 integrates phenylpropanoid metabolism and protein interaction networks in meristems to govern cold tolerance, providing reliable candidate genes and theoretical foundations for Brassica rapa cold-resistance molecular breeding.