Evolutionary diversification of the STAYGREEN gene family in Nicotiana

Chlorophyll degradation underpins leaf senescence and shapes leaf maturation and quality in Nicotiana (tobacco). This process is orchestrated by STAYGREEN (SGR) proteins that recruit chlorophyll catabolic enzymes within chloroplasts, yet how the SGR gene family has diversified and been organized in the context of polyploid Nicotiana remains largely unresolved. Here, we performed a comprehensive genome-wide and comparative analysis of SGR genes across representative monocot and dicot species, with a focus on Nicotiana. Phylogenetic analyses revealed that Nicotiana SGRs belong to a conserved angiosperm SGR lineage but exhibit copy-number expansion consistent with a polyploid genome history, with publicly available transcriptomic data further indicating expression divergence across different tissues. Codon usage analyses indicated moderate bias and predominantly mutation-driven evolution in Nicotiana SGRs, in contrast to strong GC-driven selection in grasses. Conserved motif and domain analyses demonstrated a highly constrained catalytic core and structural scaffold across species, whereas terminal regions showed lineage-specific diversification. Most Nicotiana SGR proteins retained chloroplast targeting signals, although targeting strength and cleavage-site features varied among homologs. Structural modeling further highlights the remarkable conservation of the SGR fold. Together, our findings reveal that polyploid genome evolution has expanded the Nicotiana SGR repertoire whereas preserving its core biochemical architecture, providing an evolutionary framework for understanding how chlorophyll degradation is maintained and diversified in complex plant genomes.