Evolutionary constraint on a core winter proteome across independently cold-adapted PACMAD grasses

Grasses in the Panicoideae, Aristidoideae, Chloridoideae, Micrairoideae, Arundinoideae, and Danthonioideae (PACMAD) clade independently colonized cold environments from warm-climate ancestors, but whether their molecular responses to freezing reflect shared evolutionary solutions or lineage-specific innovations remains poorly understood at the protein level. We used comparative proteomics to test whether protein-level cold responses show stronger cross-species conservation than previously observed at the transcript level. We quantified seasonal rhizome proteomes (winter vs summer) from five PACMAD species grown in a common-garden experiment exposed to sustained sub-zero temperatures, identified differentially abundant proteins, and compared fold-change magnitudes across species using orthogroup-based correlation analyses. We further examined LEA3 protein structure through hydropathy profiling and motif analysis. Shared cold-responsive proteins showed higher cross-species fold-change correlation (ρ = 0.80) than background proteins (ρ = 0.45), despite greater divergence in baseline abundance. This correlation advantage persisted (Δρ = 0.27) after controlling for effect-size selection by comparing shared cold-responsive proteins against magnitude-matched single-species responses. LEA3 was the only ortholog elevated across all five species, with cold-tolerant species containing more tandem 11-mer repeats than the cold-sensitive maize. Two species accumulated multiple LEA3 paralogs, increasing total LEA3 abundance. Independent evolution of freezing tolerance in PACMAD grasses is consistent with evolutionary constraints on protein-level response magnitude, reflecting the retention of an ancestral protective capacity. Structural divergence of LEA3 in maize suggests that transcriptional induction alone does not ensure freezing tolerance; functional protection likely requires intact motif architecture.