Date Published: January 9, 2017
Publisher: Public Library of Science
Author(s): Yemima Givaty-Rapp, Narendra Singh Yadav, Asif Khan, Gideon Grafi, Vitaly Citovsky.
Cell dedifferentiation characterizes the transition of leaf cells to protoplasts and is accompanied by global chromatin decondensation. Here we show that in Arabidopsis, chromocentric chromatin undergoes prompt and gradual decondensation upon protoplasting. We hypothesized that prompt chromatin decondensation is unlikely to be driven solely by epigenetic means and other factors might be involved. We investigated the possibility that S1-type endonucleases are involved in prompt chromatin decondensation via their capability to target and cleave unpaired regions within superhelical DNA, leading to chromatin relaxation. We showed that the expression and activity of the S1-type endonuclease 2 (ENDO2) is upregulated in dedifferentiating protoplasts concomitantly with chromatin decondensation. Mutation of the ENDO2 gene did not block or delay chromocentric chromatin decondensation upon protoplasting. Further study showed that ENDO2 subcellular localization is essentially cytoplasmic (endoplasmic reticulum-associated) in healthy cells, but often localized to the nucleus and in senescing/dying cells it was associated with fragmented nuclei. Using in gel nuclease assays we identified two ENDO2 variants, designated N1 (cytoplasmic variant) and N2 (cytoplasmic and nuclear variant), and based on their capability to bind concanavalin A (ConA), they appear to be glycosylated and de-glycosylated (or decorated with ConA non-binding sugars), respectively. Our data showed that the genome is responding promptly to acute stress (protoplasting) by acquiring decondensation state, which is not dependent on ENDO2 activity. ENDO2 undergoes de-glycosylation and translocation to the nucleus where it is involved in early stages of cell death probably by introducing double strand DNA breaks into superhelical DNA leading to local chromatin relaxation and fragmentation of nuclei.
Somatic plant cells retain their developmental capabilities and under appropriate conditions can dedifferentiated (i.e., assume stem cell like state) and give rise to different cell types that make up a new fertile plant. In plants, dedifferentiation characterizes the transition of differentiated leaf cells to protoplasts (plant cells devoid of cell walls), which is accompanied by widespread chromatin decondensation [1–3], a feature characterizing stem cells both in plants and animals [4,5]. Interestingly, somatic nuclei derived from chicken erythrocytes were induced to dedifferentiate by Xenopus egg extract, which was accompanied by prompt chromatin decondensation . This suggests that besides epigenetic means other mechanisms might be involved to bring about prompt acquisition of decondensed chromatin state. Thus, we assumed that one way by which condensed chromatin can assume a relaxed state is by nicks or double strand DNA breaks (DSBs) being introduced into superhelical DNA by the activity of S1-type single-stranded DNA endonucleases. Torsional strain generated in superhelical DNA, which is common in condensed chromatin often leads to local denaturation and unpairing , which allows single-stranded DNA endonucleases to generate nicks and/or DSBs to bring about chromatin relaxation. This is well demonstrated by the conversion of supercoiled plasmid DNA into relaxed and linear forms by S1-type endonucleases [8,9].
We showed here that chromocentric regions decondensed their chromatin promptly but gradually with pericentric decondensation occurring first within 30 min, and slightly later within 60 min, the centric region assumed open chromatin conformation. We also showed that chromatin decondensation was associated with increase in S1-type endonuclease activity driven by ENDO2, which was gradually diminished thereafter. However, we could not show a linkage between prompt chromatin decondensation and ENDO2 activity inasmuch as mutation of the ENDO2 gene did not block or delay chromocentric chromatin relaxation. Using endo2 mutant plants and in gel nuclease assays we found that the nuclease activities in protoplasts, designated N1 and N2 are contributed by ENDO2. Interestingly, ENDO2 displayed a peculiar localization in protoplasts, that is, cytoplasmic in healthy cells, often showed distinct localization within nucleoli; in senescing protoplast cells ENDO2-GFP showed association with fragmented nuclei. This mode of localization is similar to that reported for wheat endonuclease TaS1L in Arabidopsis protoplasts . Also, transient expression of BFN1/ENDO1 in tobacco protoplast cells showed cytoplasmic, ER-associated localization in normal tobacco protoplasts but localization around nuclei in senescing protoplasts . In transgenic Arabidopsis plants expressing BFN1-GFP, the protein was colocalized with fragmented nuclei in membrane-wrapped vesicles in late senescence . Furthermore, we showed that the cytoplasmic variant of ENDO2 (N1) is glycosylated and capable to bind ConA, while the nuclear variant (N2) is essentially unglycosylated or is decorated with ConA non-binding sugars.