Figure 1.
Expression patterns of Class II HDAs in different vegetative organs and developmental stages were assessed using RT-PCR with ubiquitin (UBQ) as loading control. Legend: R roots, S stem, L leaves, MS mature seeds, YRL young rosette leaf, DRL developed rosette leaf, I inflorescence, YF young flower, MF mature flower, FS flowers and siliques.
Figure 2.
Subcellular localization of Class II HDAs.
Protoplast transient expression using HDA-YFP fusion constructs were used to determine the subcellular localization of Class II HDAs. HDA5, HDA8, and HDA14 exhibited cytoplasmic localization while HDA15 concentrates inside the nucleus. VirD2NLS fused with mCherry was used as a nuclear marker. Scale bars were calibrated to 10 µm.
Figure 3.
Cell fractionation and immunoblot detection HDA-GFP transfected protoplasts were separated into cytoplasmic and nuclear fractions then subjected to immunoblot analysis using anti-GFP antibody.
Histone H3 and RHA1 were used as nuclear and cytoplasmic markers, respectively, on WT protoplasts. P protoplast extract, N nuclear fraction, C cytosolic fraction.
Figure 4.
Particle bombardment in onion epidermal tissues.
A. Onion tissues were transfected with HDA-GFP plasmids to visualize the pertinent localization of Class II HDAs. HDA5 and HDA8 both localize in the nucleus and cytoplasma while HDA14 cytoplasmic. Still, HDA15 remained nuclear. B. Onion tissues transfected with HDA5-GFP shows its localization in the cytoskeletal network with the dynamic movement of HDA5 spots (arrows) along these web-like structures. Pictures were taken at 2 sec intervals with the same onion cell.
Figure 5.
Organelle markers and Class II HDA localization.
Specific organelle markers fused with RFP and mitoTracker stain were used to identify the subcellular localization of Class II HDAs. The ER marker, HDEL, overlaps with the localization of HDA5-GFP. Although HDA8 predominantly abounds in the cytoplasm, partial nuclear localization was observed at the surrounding areas of the nucleus using VirD2NLS as nuclear marker. Moreover, HDA15 concentrates in a small spot inside the nucleus, potentially nucleolus. On the other hand, HDA14-YFP was confirmed to localize in the chloroplasts and mitochondria using mitoTracker. Protoplasts derived from Arabidopsis PSB-D lines were used for PEG transfection in HDA5, HDA8, and HDA15 while protoplasts from Arabidopsis leaves were utilized for the HDA14-YFP localization. Scale bars were calibrated to 10 µm.
Figure 6.
Interaction of HDA5 with 14-3-3.
A. HDA5 exhibited positive interaction with 14-3-3 K and ε in the cytoplasm indicating that HDA5 requires these chaperones for its nuclear export and cytoplasmic retention considering that it does not contain any nuclear export signal within its amino acid sequence. B. On the contrary, HDA15 did not elicit any interaction with 14-3-3 K nor ε suggesting that it may rely on its own nuclear localization and export signals. C. CoIP results further confirm the positive association of HDA5 with 14-3-3 K and ε. HDA5-GFP was co-transfected into protoplasts with myc-tagged 14-3-3 K or 14-3-3 ε. HDA5 was detected using anti-GFP while 14-3-3 proteins were immunoprecipitated with anti-myc monoclonal antibody. Asterisk and arrowheads indicate non-specific bands and 14-3-3-myc, respectively.
Figure 7.
Nuclear localization and export signals of HDA15.
A. Schematic representation of HDA15 is shown with its conserved histone deacetylase domain (HD), RanBP-type zinc finger (ZnF), nuclear localization signals (NLS), and nuclear export signal (NES). HDA15 contains three NLS signals, one classical par4 type NLS near the N-terminal and an overlapping bipartite NLS near the C-terminal end hereby jointly deleted as NLS2, and an NES stationed before the bipartite NLS near the carboxyl end. Numbers 1-6 illustrate the different truncated constructs of HDA15 where varying combinations of NLS and NES signals were deleted, shown here as dash lines. B. To determine if HDA15 depends on its NLS and NES for its subcellular localization, these predicted signals were truncated out of the HDA15-YFP and transiently expressed in protoplasts. Both NLS1 and NLS2 functionally direct the nuclear localization of HDA15 and remains active even in the absence of the other. However, the deletion of both NLS renders HDA15 cytoplasmic indicating the functionality of its NES. This suggests that HDA15 can translocate in and out of the nucleus and potentially undergo nucleocytoplasmic shuttling.
Figure 8.
Whole plant localization of HDA15-GFP in 4-day old seedlings.
Four day old de-etiolated seedlings of HDA15-GFP were observed for subcellular localization with the projection confocal image of the entire seedling (A), root tip (B), hypocotyl (C), and cotyledon (D). A magnified view of the leaf (E) and hypocotyl cells (F) of long day grown seedlings reveal nuclear concentrations of HDA15-GFP. On the contrary, those grown in total darkness for 4 consecutive days exhibited cytoplasmic localization of HDA15-GFP (G). In concurrence with this, transgenic protoplasts revealed nuclear confinements of HDA15-GFP upon white light treatment (E) similar to transfected protoplasts (inset E). However, 3-hour dark treated transfected protoplasts after 18 h of white light incubation resulted to its cytoplasmic translocation (H). VirD2NLS was co-transfected as nuclear marker (blue). Red color indicates autofluorescence emitted by chloroplasts. Scale bars for A to D is 100 µm, F & G was set at 25 µm, and H at 10 µm.
Figure 9.
Nucleocytoplasmic shuttling of HDA15.
To illustrate if HDA15-GFP undergoes nucleocytoplasmic shuttling, transfected protoplasts were incubated under white light overnight then covered with foil for 3 hours then re-exposed to white light. HDA15-GFP transfected protoplasts exhibited nuclear localization after 18 h of white light incubation. Further dark treatment for 3 h elicited partial cytoplasmic translocation of HDA15-GFP. Re-exposition of these protoplasts to white light after one hour lead to its complete nuclear import clearly demonstrating that light drives the nucleocytoplasmic shuttling of HDA15. VirD2NLS was co-transfected as nuclear marker (blue). Scale bars were calibrated to 10 µm.