The enigma of progressively partial endoreplication: New insights provided by flow cytometry and next-generation sequencing

Abstract

In many plant species, somatic cell differentiation is accompanied by endoreduplication, a process during which cells undergo one or more rounds of DNA replication cycles in the absence ofmitosis, resulting in nuclei withmultiples of 2C DNA amounts (4C, 8C, 16C, etc.). In some orchids, a disproportionate increase in nuclear DNA contents has been observed,where successive endoreduplication cycles result in DNA amounts 2C +P, 2C+3P, 2C+7P, etc., where P is the DNA content of the replicated part of the 2C nuclear genome. This unique phenomenonwas termed "progressively partial endoreplication" (PPE).We investigated processes behind the PPE in Ludisiadiscolor usingflowcytometry (FCM)andIllumina sequencing. Inparticular,wewanted to determinewhether chromatin elimination or incomplete genome duplication was involved, and to identify types of DNA sequences that were affected. Cell cycle analysis of root tip cell nuclei pulse-labeled with EdU revealed two cell cycles, one ending above the population of nuclei with 2C+ P content,andtheother witha typical"horseshoe" patternof S-phasenuclei rangingfrom2Cto4CDNAcontents. Theprocess leading to nuclei with 2C+P amounts therefore involves incomplete genome replication. Subsequent Illumina sequencing of flow-sorted 2C and 2C+P nuclei showed that all types of repetitiveDNAsequences were affected during PPE; a complete elimination of any specific type of repetitive DNA was not observed.We hypothesize that PPE is part of a highly controlled transition mechanism from proliferation phase to differentiation phase of plant tissue development.