Fig 1.
Chromocentric chromatin undergoes prompt and gradual decondensation upon protoplasting.
Nuclei prepared from Arabidopsis leaves (cont) or from leaves treated for 30 and 60 min with cell wall degrading enzymes were subjected to FISH using fluorescein-labeled PC-Chr1 (pericentric region) and rhodamine-labeled CEN180. Note chromatin decondensation is gradual initiating at the pericentromer followed by decondensation of the centromeric chromatin. DAPI was used as a counterstain. Bar = 5 μm.
Fig 2.
Endonuclease activity is enhanced in protoplasts immediately after preparation.
(A) ENDO2 is up-regulated in protoplasts. Based on transcriptome profiling of protoplasts obtained following 12–14 h incubation of Arabidopsis leaves with cell wall degrading enzymes [29]. Bars represent standard deviation. Inset shows RT-PCR demonstrating low and high expression of ENDO2 in leaves (L) and protoplasts (P), respectively. UBQ10 was used as a reference RNA. M, DNA size markers. (B) In vitro endonuclease conversion assay using proteins (3 μg) extracted from isolated protoplasts at various time points after preparation. Supercoiled plasmid DNA (Input, 1 μg) was incubated for 30 seconds at room temperature and DNA topology was analyzed by agarose gel electrophoresis. The positions of the different topological forms of plasmid DNA are indicated: R, relaxed, L, linear, SC, supercoiled plasmid DNA. Input is 1 μg of supercoiled plasmid DNA. Endonuclease activity in proteins extracted from green (AtGL) and yellow (AtYL) leaves was used as a reference. Note that endonuclease activity in protoplasts immediately after preparation (4h) is comparable to that of yellow, senescing leaves. (C) In-gel nuclease assay. Proteins (30μg) extracted from Arabidopsis green (AtGL) and yellow (AtYL) leaves or from protoplasts at the indicated time points after preparation were separated on polyacrylamide gel containing denatured salmon sperm DNA and subjected to in gel assay. N1, N2 and N3 nucleases are indicated by arrows.
Fig 3.
ENDO2 is modified in protoplasts.
(A) N1 and N2 nucleases are the product of the ENDO2 gene. In gel nuclease assay of proteins extracted from protoplasts derived from WT leaves (WT Col), endo2 mutant leaves and from endo2 protoplasts transformed with pUC19-S35-ENDO2-His. Note that endo2 protoplasts lack N1 and N2 nucleases. (B) ENDO2 N2 variant is not glycosylated. Proteins extracted from WT protoplasts (24h) were loaded on ConA-agarose and bound proteins were subjected to in gel nuclease assay. Note that ConA enriched the N1 and N3 nucleases. (C) ENDO2 is cleaved at its N terminus. ENDO2 was tagged with histidine (6X) at its N terminus (His-ENDO2) and the plasmid pUC19-35S-His-ENDO2 was transformed into endo2 protoplasts. His-tagged proteins extracted were purified on nickel column and bound proteins (Ni beads) as well as the flow through (FT) fraction were subjected to in gel assay. Control indicates untransformed endo2 protoplasts. (D) ENDO2 is not cleaves at its C terminus. ENDO2 was tagged with histidine (6X) at its C terminus (ENDO2-His) and the plasmid pUC19-35S-ENDO2-His was transformed into endo2 protoplasts. Bound proteins were subjected to in gel assay. Control indicates untransformed endo2 protoplasts.
Fig 4.
ENDO2 is essentially cytoplasmic in healthy cells but translocates to the nucleus in senescing, unhealthy cells.
(A) Most nuclease activity resides in the cytoplasm and a small fraction of ENDO2-N2 variant is found in the nucleus. Cytoplasmic and nuclear fractions were prepared from protoplasts at various time points after preparation and subjected to in gel nuclease assay. (B) ENDO2-GFP is associated with the ER in healthy cells. Protoplasts derived from WT leaves were cotransformed with ENDO2-GFP and ER marker CNX-RFP and analyzed under a confocal microscope after 24 h. (C) ENDO2 display various localization patterns in the nucleus of aging protoplasts. Wild type protoplasts were co-transformed with pS35-ENDO2-GFP and pS35-AtMBD5-RFP and inspected at various time points after transformation. Note the various types of ENDO2 nuclear localization and ENDO2 association with fragmented nuclei (marked with asterisks) in senescing, dying protoplasts (Type 3 and 4).
Fig 5.
Mutation of the ENDO2 gene does not block prompt chromocentric chromatin decondensation upon protoplasting.
(A) Nuclei prepared from protoplasts derived from WT and endo2 leaves treated for 30 min with cell wall degrading enzymes were subjected to FISH using rhodamine-labeled CEN180 and fluorescein-labeled PC-Chr1. (B, C) Nuclei prepared from WT leaves or from protoplasts derived from WT and endo2 leaves treated for 1 h with cell wall degrading enzymes were subjected to FISH using fluorescein-labeled CEN180 (B) and and rhodamine-labeled PC-Chr1 (C). DAPI was used as a counter stain.