The Enigma of Progressively Partial Endoreplication: New Insights Provided by Flow Cytometry and Next-Generation Sequencing
The Enigma of Progressively Partial Endoreplication: New Insights Provided by Flow Cytometry and Next-Generation Sequencing
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DOI：
10.1093/gbe/evw141
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发表时间：
2016-06-01
影响因子：
3.3
通讯作者：
Vrana, Jan
中科院分区：
文献类型：
--
作者：
Hribova, Eva;Holusova, Katerina;Vrana, Jan
在许多植物物种中，体细胞分化伴随着核内复制，这是一个细胞在没有有丝分裂的情况下经历一轮或多轮DNA复制周期的过程，导致细胞核具有2C DNA量的倍数（4C, 8C， 16C等）。在一些兰花中，已经观察到核DNA含量的不成比例的增加，其中连续的核重复循环导致DNA量2C+ P， 2C+ 3P， 2C+ 7P等，其中P是2C核基因组复制部分的DNA含量。这种独特的现象被称为“进行性局部内复制”（PPE）。我们使用流式细胞术（FCM）和Illumina测序研究了Ludisia变色PPE背后的过程。特别是，我们想确定是否涉及染色质消除或不完全基因组复制，并确定受影响的DNA序列类型。用EdU脉冲标记根尖细胞核进行细胞周期分析，发现两个细胞周期，一个结束于2C+ P含量的细胞核群体之上，另一个结束于2C - 4C DNA含量的s期细胞核的典型“马蹄形”模式。因此，导致含有2C+ P的细胞核的过程涉及不完全的基因组复制。随后对2C和2C+ P核的Illumina测序显示，PPE期间所有类型的重复DNA序列都受到了影响；没有观察到完全消除任何特定类型的重复DNA。我们假设PPE是植物组织发育从增殖阶段到分化阶段高度受控的过渡机制的一部分。
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 witha 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.