Evolution of a chloroplast subcompartment housing plastid DNA inside a cup-like starch granule

Chloroplasts evolved from a cyanobacterial endosymbiont^1,2,3,4 and have retained bacterium-derived plastid DNA (ptDNA) that is typically found in dispersed nucleoid bodies.^5,6 Here, we investigate an enigmatic chloroplast subcompartment found in the green alga Caulerpa lentillifera and several closely related species where ptDNA is contained within a single cup-like starch granule.^7,8,9 We find that starch cup size and ptDNA ploidy scale closely with the development of unusually large C. lentillifera chloroplasts, suggesting that this compartment acts to increase the upper limit on chloroplast size. The purification of the starch cups combined with protein mass spectrometry and comparative genomics identifies a family of C. lentillifera-specific chloroplast cup (CCUP) genes that originated through the fusion of sequences encoding a starch-binding (CBM20) and DNA-binding (mTERF) domain. In vitro assays show that recombinant CCUP1 CBM20 and mTERF domains bind to starch and DNA, respectively, while a full-length version can recruit DNA to the surface of starch granules. Together, these data reveal the genetic origins and biochemical activities of starch-DNA tethering proteins associated with the evolution of a starch-delimited, nucleus-like compartment for ptDNA. Due to its unique structural and molecular features, we designate this chloroplast subcompartment as the starch nucleoid body (SNB).