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. 2016 Aug;171(4):2854-68.
doi: 10.1104/pp.16.00408. Epub 2016 Jun 24.

A NAP-Family Histone Chaperone Functions in Abiotic Stress Response and Adaptation

Amit K Tripathi  1 , Ashwani Pareek  1 , Sneh Lata Singla-Pareek  2
Affiliations

A NAP-Family Histone Chaperone Functions in Abiotic Stress Response and Adaptation

Amit K Tripathi et al. Plant Physiol. 2016 Aug.

Abstract

Modulation of gene expression is one of the most significant molecular mechanisms of abiotic stress response in plants. Via altering DNA accessibility, histone chaperones affect the transcriptional competence of genomic loci. However, in contrast to other factors affecting chromatin dynamics, the role of plant histone chaperones in abiotic stress response and adaptation remains elusive. Here, we studied the physiological function of a stress-responsive putative rice (Oryza sativa) histone chaperone of the NAP superfamily: OsNAPL6. We show that OsNAPL6 is a nuclear-localized H3/H4 histone chaperone capable of assembling a nucleosome-like structure. Utilizing overexpression and knockdown approaches, we found a positive correlation between OsNAPL6 expression levels and adaptation to multiple abiotic stresses. Results of comparative transcriptome profiling and promoter-recruitment studies indicate that OsNAPL6 functions during stress response via modulation of expression of various genes involved in diverse functions. For instance, we show that OsNAPL6 is recruited to OsRad51 promoter, activating its expression and leading to more efficient DNA repair and abrogation of programmed cell death under salinity and genotoxic stress conditions. These results suggest that the histone chaperone OsNAPL6 may serve a regulatory role in abiotic stress physiology possibly via modulating nucleosome dynamics at various stress-associated genomic loci. Taken together, our findings establish a hitherto unknown link between histone chaperones and abiotic stress response in plants.

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Figures

Figure 1.
Figure 1.
Putative histone chaperone OsNAPL6 is a nuclear-localized protein possessing a functional NLS near its N terminus. Nuclear and cytoplasmic protein fractions were resolved on SDS-PAGE (12%) followed either by Coomassie/silver staining (A) or immunoblotting (B) for OsNAPL6, histone H4 (a nuclear marker), and c-FBPase (a cytosolic marker). Immunoblotting for H4 and c-FBPase confirmed the purity of the nuclear and cytoplasmic fractions, respectively. C, Fluorescence micrographs show the intracellular localization of (i) GFP, (ii) OsNAPL6-GFP (OsNAPL6 fused with GFP at its C terminus), and (iii) OsNAPL6ΔNLS-GFP (NLS-deleted OsNAPL6 fused with GFP at its C terminus) in transiently transformed onion peel epidermal cells. Bar = 20 µm. See also Supplemental Figure S1.
Figure 2.
Figure 2.
OsNAPL6 is a functional H3/H4 histone chaperone capable of assembling nucleosomes in vitro. A, A plasmid supercoiling assay was utilized for analyzing the nucleosome assembly activity of OsNAPL6 where supercoiled plasmid DNA was first relaxed with Topoisomerase I and the preincubated histone octamer-OsNAPL6 complex was added to the prerelaxed plasmid. Nucleosome-like structures were allowed to form on the relaxed plasmid template in the presence of Topoisomerase I. The proteins were digested and the DNA extracted. Supercoiling of the prerelaxed plasmid DNA resulted in topoisomers with different linking numbers. These topoisomers were separated on a 1.2% agarose gel and the gel was stained with ethidium bromide, postrunning. The positions of completely supercoiled and relaxed forms are indicated. NAB, Nucleosome assembly buffer. B to D, GST pull-down assay for analyzing the histone binding specificity of OsNAPL6. GST-OsNAPL6 fusion protein was immobilized onto glutathione-sepharose beads. Four histones in different combinations (H2A/H2B-H3/H4 [All], H3/H4 [H3 & H4], and H2A/H2B [H2A & H2B]) were added to the bead bound GST-OsNAPL6. Nonspecific binding was removed by multiple washing and the specifically bound histones were eluted. B, Input of histones used for each pull-down. C and D, The pull-down fractions were analyzed on a 15% SDS-PAGE gel. As a negative control, GST was checked for its activity to pull down the histones (C, first lane). To ensure specificity, heat denatured (hd) GST-OsNAPL6 fusion protein was analyzed for its ability to pull down the histones (C, second lane). “All” indicates all four core histones. E to H, BiFC assay was carried out to check the interaction of OsNAPL6 with various histones in planta. OsNAPL6 and H2A (E), H2B (F), H3 (G), or H4 (H) cloned in complementary split-YFP vectors were cotransformed into onion peel epidermal cells using biolistic bombardment method, as indicated. After 16 h of incubation at 28°C, the peels were stained with nuclear marker 4′,6-diamino-phenylindole (DAPI) and observed under a fluorescence microscope. Bars placed at the bottom of the micrographs represents 20 μm. BF, Bright field. See also Supplemental Figure S2.
Figure 3.
Figure 3.
OsNAPL6 expression improves tolerance toward multiple abiotic stresses. A, Leaf strip senescence assay to analyze the response of OsNAPL6-overexpression and -knockdown toward salinity, dehydration, osmotic, and oxidative stresses. The assay was carried out to test the ability of OsNAPL6-Ox and -KD plants to resist loss in chlorophyll under salinity (200 mm NaCl), dehydration (5% PEG), osmotic (500 mm mannitol), and oxidative (5 mm H2O2) stress conditions. Leaf strips were floated on either water (control) or solutions containing stress agents as indicated. Numbers at the top indicate transgenic line number. Senescence was assessed until 120 h of stress. Leaf strips from wild-type, OsNAPL6-KD, and -Ox plants subjected to leaf strip senescence assay were used for estimation of either total chlorophyll (B) or chlorophyll-to-carotenoid ratio (C). FW, Fresh weight. Data shown are mean ± sd; n = 3. The * and ** represent statistically significant difference (compared to the wild type under the same conditions) at P < 0.05 and P < 0.01, respectively. Statistical significance was tested by one-way ANOVA followed by posthoc comparisons using Tukey-Kramer test.
Figure 4.
Figure 4.
Morphology of wild-type, OsNAPL6-Ox, and OsNAPL6-KD rice plants under salinity stress and after recovery from drought stress conditions. A, Approximately 2-month-old wild-type, OsNAPL6-Ox (lines Ox2.4 and Ox3.2), and OsNAPL6-KD (KD1.2) rice plants were maintained under control conditions by irrigating with water. Similar set of plants were subjected to salinity stress by fortnightly irrigation with a mixture of salt solutions leading to soil electrical conductivity (EC) of 10 dS/m (B) and drought stress (C). For drought stress, water was withheld for 12 d followed by rewatering. Growth of plants was monitored and pictures were taken on days as indicated. D, Leaves from salinity treated plants were harvested 15 d after stress and Na+/K+ ratio was determined by atomic absorption spectroscopy. E, RWC as measured 15 d postsalinity stress and 3 d postrecovery after drought stress. Data shown are mean ± sd; n = 3. The * and ** represent statistically significant difference (compared to the wild type under the same condition) at P < 0.05 and P < 0.01, respectively (one-way ANOVA followed by posthoc comparisons using Tukey-Kramer test).
Figure 5.
Figure 5.
Evaluation of various physiological and agronomic parameters shows positive correlation between OsNAPL6 expression and adaptation to salinity and drought stress. Approximately 2-month-old wild-type, OsNAPL6-Ox (lines Ox2.4 and Ox3.2), and OsNAPL6-KD (KD1.2) rice plants were subjected to salinity and drought stress. After 15 d of salinity stress (A) and 3 d postrecovery from drought stress (B), various physiological parameters, viz. net photosynthetic rate (NPR), Fv/Fm, stomatal conductance, transpiration rate, and electron transport rate (ETR), were measured and the relative values (wild-type control, taken as 100%) were plotted as a web diagram. Plants irrigated with water served as controls. After maturity, total biomass (C) and various yield-associated parameters viz. number of panicles (D), filled grains per plant (E), and harvest index (F) were determined and plotted as bar graphs. Data shown are mean ± sd; n = 3. The * and ** represent statistically significant difference (compared to the wild type under the same condition) at P < 0.05 and P < 0.01, respectively. Statistical significance was tested by one-way ANOVA followed by posthoc comparisons using Tukey-Kramer test.
Figure 6.
Figure 6.
Manipulating OsNAPL6 expression by overexpression and knockdown causes large-scale changes in the rice transcriptome. A, Number of genes up-regulated (green) and down-regulated (red) in Ox plants with respect to wild-type plants. B, Number of genes up-regulated (green) and down-regulated (red) in KD plants with respect to wild-type plants. C, Venn diagram of genes showing up-regulation in Ox plants and down-regulation in KD plants with respect to wild-type plants. Genes common to both the categories and thus showing reciprocal expression pattern are represented by the intersection. D, Venn diagram of genes getting downregulated in Ox plants and upregulated in KD plants with respect to wild-type plants. Genes common to both the categories and thus showing reciprocal expression pattern are represented by the intersection. E, qRT-PCR based validation of the microarray-based expression profile of 14 genes functioning in DNA recombination or repair, transcription, or stress response. Heat maps represent fold change in expression (in log2 scale) of OsNAPL6-Ox and -KD seedlings over wild-type seedlings and were generated using Euclidean distance-based hierarchical clustering. For this, seeds of wild-type, Ox, and KD were germinated and 12-d-old seedlings were used to isolate RNA followed by qRT-PCR. Locus ids of the genes are given in Supplemental Table S1. See also Supplemental Figure S8.
Figure 7.
Figure 7.
ChIP-qPCR to analyze the recruitment of OsNAPL6 onto the promoter regions of OsDREB1C, OsRad51, and OsLEA9. Recruitment of OsNAPL6 onto the promoter regions of OsDREB1C, OsRad51, and OsLEA9 was analyzed by ChIP-qPCR. For this, seeds of wild-type, OsNAPL6-Ox, and OsNAPL6-KD plants were germinated. Twelve-day-old seedlings were then subjected to osmotic stress (500 mm mannitol) for 8 h. Both stressed and unstressed (control) whole seedlings were harvested and subjected to ChIP (using anti-OsNAPL6 antibody). The presence of DNA regions of interest (part of the promoter region [up to 400–500 bp upstream]) of OsDREB1C (A and B), OsRad51 (C and D), and OsLEA9 (E and F) was analyzed by qPCR using the immunoprecipitated DNA after elution. The recruitment is expressed as fold enrichment over IgG (mock). Data shown are mean ± se; n = 3.
Figure 8.
Figure 8.
TUNEL assay to detect in situ DNA fragmentation in root tips of wild-type, OsNAPL6-Ox, and OsNAPL6-KD rice seedlings under control, salinity, and genotoxic stress conditions. To detect in situ DNA fragmentation in root tips, seeds from wild-type, OsNAPL6-Ox, and OsNAPL6-KD plants were germinated and grown on 0.5× MS + solid agar medium for 12 d and then subjected to 200 mm NaCl (for salinity stress) for 48 h (B) or to genotoxic stress by using 40 mg/L aphidicolin for 48 h (C) followed by fixing, permeabilization, and TUNEL reaction. Water-treated seedlings served as controls (A). Green color represents fluorescein-12-dUTP, while blue color shows DAPI staining. For microscopy, samples were mounted on slides in ProLong Gold Antifade mountant with DAPI (Life Technologies) and the slides were viewed under a confocal microscope (Nikon A1R) using a 20× objective. Images were acquired and processed using NIS Elements software (Nikon). Bar = 100 µm. BF, Bright field; F, fluorescein (green).
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