The Enigma of Progressively Partial Endoreplication: New Insights Provided by Flow Cytometry and Next-Generation Sequencing.
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 of mitosis, resulting in nuclei with multiples 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 phenomenon was termed "progressively partial endoreplication" (PPE). We investigated processes behind the PPE in Ludisia discolor using flow cytometry (FCM) and Illumina sequencing. In particular, we wanted to determine whether 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, and the other with a typical "horseshoe" pattern of S-phase nuclei ranging from 2C to 4C DNA contents. The process 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 repetitive DNA sequences 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.
Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Olomouc, Czech Republic.
Eva
E
Hřibová
Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Olomouc, Czech Republic.
Kateřina
K
Holušová
Institute of Botany, the Czech Academy of Sciences, Průhonice, Czech Republic Department of Botany, Faculty of Science, Charles University in Prague, Czech Republic Biotechnological Centre, Faculty of Agriculture, University of South Bohemia in České Budějovice, Czech Republic.
Pavel
P
Trávníček
Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Olomouc, Czech Republic.
Beáta
B
Petrovská
Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Czech Republic Prague Botanical Garden, Prague, Czech Republic.
Jan
J
Ponert
Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Olomouc, Czech Republic.
Hana
H
Šimková
Biotechnological Centre, Faculty of Agriculture, University of South Bohemia in České Budějovice, Czech Republic.
Barbora
B
Kubátová
Department of Ecosystem Biology, Faculty of Science, University of South Bohemia in České Budějovice, Czech Republic.
Jana
J
Jersáková
Biotechnological Centre, Faculty of Agriculture, University of South Bohemia in České Budějovice, Czech Republic.
Vladislav
V
Čurn
Institute of Botany, the Czech Academy of Sciences, Průhonice, Czech Republic Department of Botany, Faculty of Science, Charles University in Prague, Czech Republic.
Jan
J
Suda
Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Olomouc, Czech Republic.
Jaroslav
J
Doležel
Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, Olomouc, Czech Republic vrana@ueb.cas.cz.
Jan
J
Vrána
D002467:Cell Nucleus / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D062951:Endoreduplication / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods*; D018745:Genome, Plant; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008938:Mitosis / Q000235:genetics; D029595:Orchidaceae / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics; D011123:Polyploidy
DNA replication; EdU; Ludisia discolor; cell cycle; endoreduplication; orchids