Alternative Promoters Expand Transcriptional Complexity of Temperature Stress Responses in Cassava

Plant abiotic stress responses involve two major gene expression regulatory mechanisms: alternative promoter usage and differential expression. Although differentially expressed genes (DEGs) have been extensively studied, alternative promoter genes (APGs) remain poorly characterized despite their potential importance. We systematically compared APGs and DEGs in cassava (Manihot esculenta), a major crop tolerant to heat, but sensitive to cold. On the basis of a high-resolution transcription start site analysis (nAnT-iCAGE-seq), we identified 1,705 heat- responsive and 491 cold-responsive APGs as well as 4,172 heat-responsive and 582 cold-responsive DEGs. We identified three key distinctions. First, APGs contain fewer upstream open reading frames (uORFs) than DEGs and result in proteins with a truncated N-terminal, leading to altered subcellular localization, especially from plastids to the cytoplasm and from the cytoplasm to the nucleus. Second, while DEGs are highly conserved between cassava and Arabidopsis thaliana, the number of APGs increased in a lineage-specific manner through recent gene duplication events. Third, these duplications enabled the selective modification of cis-regulatory elements, contributing to increased APG expression (relative to DEG expression). Our findings suggest alternative promoters are essential components of the regulatory mechanism underlying the diversity in protein localization during plant stress responses, while also complementing well-characterized DEG-related stress responses.