Although several resurrection plant genomes have been sequenced, the lack of suitable dehydration-sensitive outgroups has limited genomic insights into the origin of desiccation tolerance. Here, we utilized a comparative system of closely related desiccation-tolerant (Lindernia brevidens) and -sensitive (Lindernia subracemosa) species to identify gene- and pathway-level changes associated with the evolution of desiccation tolerance. The two high-quality Lindernia genomes we assembled are largely collinear, and over 90% of genes are conserved. L. brevidens and L. subracemosa have evidence of an ancient, shared whole-genome duplication event, and retained genes have neofunctionalized, with desiccation-specific expression in L. brevidens. Tandem gene duplicates also are enriched in desiccation-associated functions, including a dramatic expansion of early light-induced proteins from 4 to 26 copies in L. brevidens. A comparative differential gene coexpression analysis between L. brevidens and L. subracemosa supports extensive network rewiring across early dehydration, desiccation, and rehydration time courses. Many LATE EMBRYOGENESIS ABUNDANT genes show significantly higher expression in L. brevidens compared with their orthologs in L. subracemosa. Coexpression modules uniquely upregulated during desiccation in L. brevidens are enriched with seed-specific and abscisic acid-associated cis-regulatory elements. These modules contain a wide array of seed-associated genes that have no expression in the desiccation-sensitive L. subracemosa. Together, these findings suggest that desiccation tolerance evolved through a combination of gene duplications and network-level rewiring of existing seed desiccation pathways.

Reproductive structures of plants (e.g. seeds) and vegetative tissues of resurrection plants can tolerate desiccation. Many genes encoding desiccation-related proteins (DRPs) have been identified in the resurrection plant Craterostigma plantagineum, but the function of these genes remains mainly hypothetical. Here, the importance of the DRP gene pcC13-62 for desiccation tolerance is evaluated by analysing its expression in C. plantagineum and in the closely related desiccation-tolerant species Lindernia brevidens and the desiccation-sensitive species Lindernia subracemosa. Quantitative analysis revealed that pcC13-62 transcripts accumulate at a much lower level in desiccation-sensitive species than in desiccation-tolerant species. The study of pcC13-62 promoters from these species demonstrated a correlation between promoter activity and gene expression levels, suggesting transcriptional regulation of gene expression. Comparison of promoter sequences identified a dehydration-responsive element motif in the promoters of tolerant species that is required for dehydration-induced β-glucuronidase (GUS) accumulation. We hypothesize that variations in the regulatory sequences of the pcC13-62 gene occurred to establish pcC13-62 expression in vegetative tissues, which might be required for desiccation tolerance. The pcC13-62 promoters could also be activated by salt stress in Arabidopsis thaliana plants stably transformed with promoter::GUS constructs.